Patent Application
20220268785
The field of the invention relates to compositions and methods for performing a mass spectrometry-based proteomic analysis of proteome and target proteins with genetic alterations and post-translational modifications. In particular, the field of the invention relates to compositions and methods for preparing a sample for one-pot quantitative proteomics near and at single-cell and subcellular levels with minimal protein loss and maximal protein recovery during processing, and for global proteome profiling and targeted analyses of peptide variants and mutations in normal and abnormal cells (such as cancer) via mass spectrometry.
In one aspect of the current disclosure, methods for performing proteomic analysis on a sample are provided. In some embodiments, the methods comprise treating the sample with a non-ionic surfactant, performing mass spectrometry on the treated sample, and detecting proteins in the treated sample. In some embodiments, the non-ionic surfactant is an alkyl glucoside. In some embodiments, the non-ionic surfactant is an alkyl diglucoside. In some embodiments, the non-ionic surfactant is an alkyl maltoside. In some embodiments, the non-ionic surfactant is octyl-maltoside, decyl-maltoside, dodecyl-maltoside, or tetradecyl-maltoside. In some embodiments, the non-ionic surfactant is n-dodecyl-β-D-maltoside (In some embodiments, the concentration is 0.01% to 0.02%. In some embodiments, the concentration is 0.015%. In some embodiments, the method results in at least about a 20-fold enhancement in the mass spectrometry signal from the sample when compared to a sample not treated with the non-ionic surfactant. In some embodiments, the detected protein comprises an amino acid sequence of a peptide of Tables 2-7.
In another aspect of the current disclosure, methods for performing proteomic analysis on a single cell are provided. In some embodiments, the methods comprise isolating a single cell to prepare a sample, treating the sample with a non-ionic surfactant, performing mass spectrometry on the treated sample and detecting proteins in the treated sample. In some embodiments, the non-ionic surfactant is an alkyl glucoside. In some embodiments, the non-ionic surfactant is an alkyl diglucoside. In some embodiments, the non-ionic surfactant is an alkyl maltoside. In some embodiments, the non-ionic surfactant is octyl-maltoside, decyl-maltoside, dodecyl-maltoside, or tetradecyl-maltoside. In some embodiments, the non-ionic surfactant is n-dodecyl-β-D-maltoside (DDM). In some embodiments, the concentration of the non-ionic surfactant is 0.005% to 0.1%. In some embodiments, the concentration is 0.01% to 0.02%. In some embodiments, the concentration is 0.015%. In some embodiments, the method results in at least about a 20-fold enhancement in the mass spectrometry signal from the sample when compared to a sample not treated with the non-ionic surfactant. In some embodiments, the detected protein comprises an amino acid sequence of a peptide of Tables 2-7.
As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.
As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of” should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.
The phrase “such as” should be interpreted as “for example, including.” Moreover, the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.
Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”
All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.
The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”
The phrases “% sequence identity,” “percent identity,” or “% identity” refer to the percentage of amino acid residue matches between at least two amino acid sequences aligned using a standardized algorithm. Methods of amino acid sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail below, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. Percent identity for amino acid sequences may be determined as understood in the art. (See, e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastp,” that is used to align a known amino acid sequence with other amino acids sequences from a variety of databases.
The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. A protein may comprise different domains, for example, a nucleic acid binding domain and a nucleic acid cleavage domain. In some embodiments, a protein comprises a proteinaceous part, e.g., an amino acid sequence constituting a nucleic acid binding domain.
Nucleic acids, proteins, and/or other compositions described herein may be purified. As used herein, “purified” means separate from the majority of other compounds or entities, and encompasses partially purified or substantially purified. Purity may be denoted by a weight by weight measure and may be determined using a variety of analytical techniques such as but not limited to mass spectrometry, HPLC, etc.
Polypeptide sequence identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
The terms “nucleic acid” and “nucleic acid molecule,” as used herein, refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Nucleic acids generally refer to polymers comprising nucleotides or nucleotide analogs joined together through backbone linkages such as but not limited to phosphodiester bonds. Nucleic acids include deoxyribonucleic acids (DNA) and ribonucleic acids (RNA) such as messenger RNA (mRNA), transfer RNA (tRNA), etc. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides are linear molecules, in which adjacent nucleotides are linked to each other via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g. nucleotides and/or nucleosides). In some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms “oligonucleotide” and “polynucleotide” can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, “nucleic acid” encompasses RNA as well as single and/or double-stranded DNA. Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule may be a non-naturally occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA/RNA hybrid, or include non-naturally occurring nucleotides or nucleosides. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and/or similar terms include nucleic acid analogs, i.e. analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in the 5′ to 3′ direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g. adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadeno sine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages).
The term “hybridization”, as used herein, refers to the formation of a duplex structure by two single-stranded nucleic acids due to complementary base pairing. Hybridization can occur between fully complementary nucleic acid strands or between “substantially complementary” nucleic acid strands that contain minor regions of mismatch. Conditions under which hybridization of fully complementary nucleic acid strands is strongly preferred are referred to as “stringent hybridization conditions” or “sequence-specific hybridization conditions”. Stable duplexes of substantially complementary sequences can be achieved under less stringent hybridization conditions; the degree of mismatch tolerated can be controlled by suitable adjustment of the hybridization conditions. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length and base pair composition of the oligonucleotides, ionic strength, and incidence of mismatched base pairs, following the guidance provided by the art (see, e.g., Sambrook et al., 1989, Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Wetmur, 1991, Critical Review in Biochem. and Mol. Biol. 26(3/4):227-259; and Owczarzy et al., 2008, Biochemistry, 47: 5336-5353, which are incorporated herein by reference).
The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and/or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter. The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof, as well as additional elements. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of” and “consisting of” those certain elements.
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.
No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and/or description found in the cited references.
The field of single-cell proteomic analysis of regular-size mammalian cells remains highly challenging, primarily due to technical difficulties in effective sampling and processing. In particular, protein loss due to adsorption remains a major pitfall of any small sample size proteomics methodology, e.g., single-cell proteomics. To alleviate the shortcomings of existing proteomic approaches, the inventors developed a broadly adoptable MS method for quantitative single-cell proteomics for both label-free and tandem mass tag (TMT) labeling analysis. This method capitalizes on surfactant-assisted one-pot (single tube or multi-well plate) processing coupled with MS (termed SOP-MS) for greatly reducing the surface adsorption losses, thus, improving detection sensitivity for MS analysis of single cells and mass-limited clinical specimens. Critically, the inventors discovered that the use of alkly glucosides, e.g., n-Dodecyl β-D-maltoside (DDM), maximizes recovery for quantitative small sample proteomics by greatly reducing surface adsorption losses.
As used herein, “n-Dodecyl β-D-maltoside (DDM)” refers to a compound with a formula:
Accordingly, in one aspect of the current disclosure, methods for performing proteomic analysis on a sample are provided. In some embodiments, the methods comprise treating the sample with a non-ionic surfactant, performing mass spectrometry on the treated sample, and detecting proteins in the treated sample.
As used herein, “detecting” refers to determining the presence of a protein, or a portion thereof in a sample. In some embodiments, detecting comprises determining the presence of a peptide, wherein a protein comprises said peptide; Thus, detection of the peptide may confirm the presence of a protein in the sample. For example, detection of the peptide with sequence consisting of SEQ ID NO:1, which is an oncogenic variant derived from KRAS, indicates the presence of KRAS, i.e., oncogenic mutant KRAS, in a sample. Similarly, detection of a variety of proteins can be accomplished by detecting a peptide with an amino acid sequence of a peptide found in Tables 2-7. In some embodiments, the peptide found in a table is described by a variant, or “non-canonical” amino acid, inserted amino acid, or deleted amino acid. It will be apparent to one of skill in the art that such information can be used to describe peptides that are detected by the disclosed methods by referring to the canonical sequence of the given protein and making the change to the amino acid sequence that is indicated in the table. In some embodiments, detection is automated and does not require the use of the human mind.
In some embodiments, the non-ionic surfactant is an alkyl glucoside. In some embodiments, the non-ionic surfactant is an alkyl diglucoside. In some embodiments, the non-ionic surfactant is an alkyl maltoside. In some embodiments, the non-ionic surfactant is octyl-maltoside, decyl-maltoside, dodecyl-maltoside, or tetradecyl-maltoside. In some embodiments, the non-ionic surfactant is n-dodecyl-β-D-maltoside (DDM). In some embodiments, the concentration of the non-ionic surfactant is 0.005% to 0.1%. In some embodiments, the concentration is 0.01% to 0.02%. In some embodiments, the concentration is 0.015%. In some embodiments, the method results in at least about a 20-fold enhancement in the mass spectrometry signal from the sample when compared to a sample not treated with the non-ionic surfactant. In some embodiments, the detected protein comprises an amino acid sequence of a peptide of Tables 2-7.
As used herein, “proteomic analysis” refers to any technique whereby the proteome, or a portion thereof, of a sample from a subject is sequenced. In some embodiments, sequencing comprises determining the amino acid sequence of proteins in a sample. In some embodiments, sequencing comprises determining a substantial portion of the amino acid sequences of proteins in a sample, e.g., sequencing 50% of the proteins, 60% of the proteins, 70% of the proteins, 80% of the proteins, 90% of the proteins, 95% of the proteins, or more than 95% of the proteins in a sample. In some embodiments, proteomic analysis comprises mass spectrometry. In some embodiments, proteomic analysis comprises liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS or LC-MS2), which may, in some embodiments, includes high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS).
Mass spectrometry is an analytical technique used to measure the mass-to-charge ratio (m/z or m/q) of ions. It is most generally used to analyze the composition of a physical sample by generating a mass spectrum representing the masses of sample components. The technique has several applications including identifying unknown compounds by the mass of the compound and/or fragments thereof determining the isotopic composition of one or more elements in a compound, determining the structure of compounds by observing the fragmentation of the compound, quantitating the amount of a compound in a sample using carefully designed methods (mass spectrometry is not inherently quantitative), studying the fundamentals of gas phase ion chemistry (the chemistry of ions and neutrals in vacuum), and determining other physical, chemical or even biological properties of compounds with a variety of other approaches.
A mass spectrometer is a device used for mass spectrometry, and it produces a mass spectrum of a sample to analyze its composition. This is normally achieved by ionizing the sample and separating ions of differing masses and recording their relative abundance by measuring intensities of ion flux. A typical mass spectrometer comprises three parts: an ion source, a mass analyzer, and a detector.
The kind of ion source is a contributing factor that strongly influences-what types of samples can be analyzed by mass spectrometry. Electron ionization and chemical ionization are used for gases and vapors. In chemical ionization sources, the analyte is ionized by chemical ion-molecule reactions during collisions in the source. Two techniques often used with liquid and solid biological samples include electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). Other techniques include fast atom bombardment (FAB), thermospray, atmospheric pressure chemical ionization (APCI), secondary ion mass spectrometry (SIMS), and thermal ionisation.
Liquid-chromatography-tandem-mass spectrometry (LC-MS/MS) has been introduced in clinical chemistry (Vogeser M., Clin. Chem. Lab. Med. 41 (2003) 117-126). Advantages of this technology are high analytical specificity and accuracy and the flexibility in the development of reliable analytical methods. In contrast to gas chromatography mass spectrometry (GC-MS) as the traditional mass spectrometric technology in clinical chemistry. LC-MS/MS has been shown to be a robust technology, allowing its application also in a large-scale routine laboratory setting.
The inventors demonstrated that treating of samples for proteomic analysis, e.g., LC-MS/MS, with DDM decreases the loss of proteins due to adsorption to surfaces used in handling and preparing the samples, e.g., tubes, plates, etc. Therefore, inclusion of DDM in the preparation of samples for proteomic analysis, e.g., LC-MS/MS increases the mass spectrometry signal at least about 20-fold than without treatment with DDM (
A key factor for development of single-cell proteomic assays is the ability to preserve the small amount of starting material derived from a sample consisting of one or a small number of cells. As used herein, a “small number of cells” is less than 50 cells, less than 40 cells, less than 30 cells, less than 20 cells, preferably less than 10 cells. Thus, the inventors demonstrated that treatment of small numbers of cell samples with DDM allows detection of protein variants, e.g., oncogenic variants (
Therefore, in some embodiments, methods for performing proteomic analysis on a single cell are provided. In some embodiments, the methods comprise isolating a single cell to prepare a sample, treating the sample with a non-ionic surfactant, and performing mass spectrometry on the treated sample. In some embodiments, the methods comprise isolating a single cell to prepare a sample, treating the sample with a non-ionic surfactant, performing mass spectrometry on the treated sample and detecting proteins in the treated sample. In some embodiments, the non-ionic surfactant is an alkyl glucoside. In some embodiments, the non-ionic surfactant is an alkyl diglucoside. In some embodiments, the non-ionic surfactant is an alkyl maltoside. In some embodiments, the non-ionic surfactant is octyl-maltoside, decyl-maltoside, dodecyl-maltoside, or tetradecyl-maltoside. In some embodiments, the non-ionic surfactant is n-dodecyl-β-D-maltoside (DDM). In some embodiments, the concentration of the non-ionic surfactant is 0.005% to 0.1%. In some embodiments, the concentration is 0.01% to 0.02%. In some embodiments, the concentration is 0.015%. In some embodiments, the method results in at least about a 20-fold enhancement in the mass spectrometry signal from the sample when compared to a sample not treated with the non-ionic surfactant. In some embodiments, the detected protein comprises an amino acid sequence of a peptide of Tables 2-7.
The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.
The disclosed subject matter relates to a methodology breakthrough with a 20-fold improvement of sample recovery for mass spectrometry-based single-cell proteomic analyses.
Large numbers of cells are generally required for quantitative global proteome profiling due to the significant surface adsorption losses associated with sample processing. Such bulk measurement obscures important cell-to-cell variability (cell heterogeneity) and makes proteomic profiling impossible for rare cell populations, such as circulating tumor cells (CTCs) and early metastatic cells. Herein the inventors report a facile mass spectrometry (MS)-based single-cell proteomics method that capitalizes on a MS-compatible nonionic surfactant, n-Dodecyl-β-D-maltoside (DDM), for greatly reducing the surface adsorption losses by ˜20-fold for effective single-tube processing of single cells, thus significantly improving detection sensitivity for single-cell proteomic analysis. With standard MS platforms, the method allows for the first time precise, label-free, reliable quantification of hundreds of proteins from single human cells in a simple, convenient manner. When applied to a patient CTC-derived xenograft (PCDX) model, the method can reveal distinct protein signatures between primary tumor cells and early metastases to the lungs at the single-cell resolution. The approach paves the way for routine, precise quantitative single-cell proteomic analysis.
Applications
The disclosed subject matter has applications which may include, but are not limited to: (i) both global and targeted single-cell proteomics in all biomedical fields; (ii) elucidation of cellular heterogeneity across and within populations, especially rare populations of stem cells, circulating tumor cells, and early metastatic cells; (iii) potential applications to subcellular organelle proteomics, like nucleus, mitochondria, etc.; and (iv) 3D or 4D proteomic mapping of normal and pathological tissues at single cell resolution.
Advantages
The disclosed subject matter has advantages which may include, but are not limited to the following. There is no exisiting commercial services for single-cell proteomics due to technical barriers from surface adsorption losses duing sample processing. Previous two single-cell proteomic methods based on nanoPOTS-Lumos MS1 and iPAD1-Lumos MS2 require specific device and are extremely difficult for broad dissemination.
The disclosed breakthrough methodology based on the nonionic surfactant DDM additive increases 20-fold in sample recovery and compatible with standard mass-spectrometry for convenient commercialization. Moreover, the sample recovery and peptide analyses are similar to that with two previous methods on special devices. The broad applications of incoming single-cell proteomic analyses will bring unprecedented impact to the biological and medical field, including basic science, translational research, and clinical medicine.
To alleviate the shortcomings of existing proteomic approaches, the inventors have recently developed a facile, broadly adoptable MS method for precise quantitative single-cell proteomic analysis. This method capitalizes on surfactant-assisted one-pot processing coupled with MS (termed SOP-MS) for greatly reducing the surface adsorption losses, thus significantly improving detection sensitivity for MS analysis of single cells. SOP-MS was demonstrated to enable reliable quantification of hundreds of proteins from single cells with standard MS platforms. When it was applied to analyze two types of single cells isolated from patient CTC-derived xenografts (PCDXs): CTCs propagated in the mouse mammary fat pads with CSC properties (primary tumor cells) and their early micrometastases seeded to the lungs (lung micromets), SOP-MS not only allows for identification of protein signatures that can be leveraged for CTC characterization, but also facilitates elucidating heterogeneous alterations of metastatic tumor cells upon colonization of the lungs. Interestingly, the protein alterations in these cells are related to the selection pressure of anti-tumor immunity (e.g., neutrophils and innate immunity) for the transition from primary tumor CTCs to the early metastatic cells. These results demonstrate great potential of SOP-MS for broad applications in quantitative single-cell proteomics.
Recent advances in nucleic acid amplification-based sequencing technologies allow for comprehensive characterization of genome and transcriptome in single mammalian or tumor cells1-3. Since no protein amplification methods exist for single cell proteome profiling, current single-cell proteomics technologies primarily rely on antibody-based immunoassays (e.g., mass cytometry) for targeted measurements4, but they share the limitations of antibody-based approaches5. Mass spectrometry (MS)-based proteomics is a promising alternative for quantitative single-cell proteomics because it is antibody-free and has high specificity and ultrahigh multiplexing capability6. Sophisticated sample preparation methods are generally used to process standard proteomics samples with large amounts of starting materials (e.g., ≥1000 ug or ≥10 million human cells) for comprehensive proteomic analysis7-10. However, they cannot be used to process smaller samples (e.g., low μg or sub-μg levels of starting materials). With this recognition, in the past decade great efforts have been made for effective processing of smaller samples using single-pot sample preparation (e.g., in-StageTip11, 12 and SP313, 14) and immobilized enzyme processing systems (e.g., IMER15, 16 and SNaPP17). Using the in-StageTip device combined with Tip-based sample fractionation, >7000 proteins across 12 immune cell types were reported when ˜15,000 immune cells (˜2 μg) were analyzed12. The SP3 protocol can allow reproducible quantification of 500-1000 proteins from 100-1000 HeLa cells14. With improved sample processing as well as recent advances in detection sensitivity, MS-based single-cell proteomics has recently been used for deep proteome profiling of large-size single cells (e.g., oocytes and blastomeres at ˜0.1-100 μg of protein amount per cell)13, 18-20. However, single-cell proteomic analysis of regular-size mammalian cells (typically ˜100 μg per cell) remains highly challenging, primarily due to technical difficulties in effective sampling and processing21-23. In recent three years great progress has been made to improve processing recovery from low numbers of cells by either reducing sample processing volume (e.g., nanoPOTS, OAD, and iPAD-1 devices downscaling the processing volume to ˜2-200 nL for label-free global proteomics21, 24, 25) or using excessive amounts of carrier proteins or proteome (e.g., the addition of exogenous BSA as a carrier protein for targeted proteomics22, 23 or tandem mass tag (TMT)-labeled 100s of cells as a carrier channel for TMT labeling-based global proteomics26). However, all these approaches have technical drawbacks: nanoPOTS, OAD, and iPAD-1 are not easily adoptable for broad benchtop applications21, 24, 25; exogenous protein carrier is more suitable for targeted proteomics. have; a TMT carrier is added after sample processing, and thus it cannot effectively prevent the surface adsorption losses during initial sample processing26, resulting in low reproducibility with a correlation coefficient of only ˜0.2-0.4 between replicates for ineffectively processed single cells27. Furthermore, due to the inability to fractionate ultrasmall TMT carrier samples, TMT labeling-based global proteomics suffers from ratio compression or distortion caused by coeluting interferences28. Therefore, only three MS-based single-cell proteomics methods are available for reliable label-free analysis of regular-size single mammalian cells, but they need specific devices and/or a skilled person to operate which limit their potential for wide adoptions by research community.
Single-cell proteomics can empower characterization of cell functional heterogeneity and reveal important protein signatures at the single-cell level for rare cell populations, such as cancer stem cells, circulating tumor cells (CTCs), and early metastatic cells. When compared to peripheral blood mononuclear cells (PBMCs), CTCs are rare (normally less than 0.1%). Their seeding efficiency is extremely low but CTCs with stem cell properties can cluster and colonize at relatively high efficiency29-33. CTCs can remain in the blood stream for up to several hours as single cells or tumor clusters, and sometimes they associate with various other cell types (e.g., neutrophils) until they extravasate at a potential site of metastasis29, 34-36. However, there are no available tools for proteomic characterization of CTCs that can elucidate their heterogeneity as well as dynamic alterations upon formation of early micrometastases. Therefore, it still remains uncertain whether metastatic tumor cells undergo an epithelial to mesenchymal transition (EMT) and/or a mesenchymal-to-epithelial transition (MET) at metastatic seeding37-40.
To alleviate the shortcomings of existing proteomic approaches, the inventors have recently developed a broadly adoptable MS method for quantitative label-free single-cell proteomic analysis. This method capitalizes on surfactant-assisted one-pot (single tube or multi-well plate) processing coupled with MS (termed SOP-MS) for greatly reducing the surface adsorption losses, thus improving detection sensitivity for MS analysis of single cells and mass-limited clinical specimens (
Results
‘All-In-One’ SOP-MS for Maximizing Single-Cell Recovery
The major issue for current MS-based bottom-up single-cell proteomics is substantial surface adsorption losses. Proteins are ‘stickier’ than other biomolecules (e.g., nucleic acids) and need to be digested into peptides for efficient MS analysis which involves multistep sample processing. Both BSA and surfactants are commonly used as additives to minimize surface adsorption for low amounts of proteins and peptides. Unfortunately, the addition of BSA is not suitable for label-free single-cell global proteomics analysis22, 23. Most ionic surfactants (e.g., sodium dodecyl sulfate) are not MS-compatible and require multiple cleanup steps that cause substantial sample loss, especially for small numbers of cells, though they are highly efficient for cell lysis and protein denaturation41. Nonionic surfactants are known to substantially reduce protein adsorption for hydrophobic surface-based vessels (e.g., single tube or single well) while they have less effects on hydrophilic surfaces (e.g., glass vials), because they have much stronger binding strength than proteins for the hydrophobic surface. They are broadly used to modulate protein aggregation, adsorption loss, stability, and activity in pharmaceutical and biotechnology industries. However, most nonionic surfactants (e.g., octylglucoside) are coeluted with tryptic peptides, which severely affects peptide detection due to ionization suppression42.
n-Dodecyl β-D-maltoside (DDM), a classic nonionic surfactant, is an exception. It has been demonstrated to robustly solubilize membrane proteins for effective cell lysis43, 44, and to be highly compatible with MS without requiring surfactant removal and is eluted at a high percentage of organic solvent where it does not impact peptide detection43, 44. Furthermore, DDM is sufficiently thermostable to tolerate the high temperature used for cell lysis and protein denaturation, and can also enhance trypsin and Lys-C enzyme activity42. Therefore, the inventors have recently developed a nonionic surfactant DDM-assisted one-pot sample preparation coupled with MS termed SOP-MS that combines all steps into one pot (e.g., single PCR tube or single well from a multi-well PCR plate routinely used for single-cell genomics and transcriptomics) including single-cell collection, multistep single-cell processing, and elimination of all transfer steps with direct sample loading for LC-MS analysis (
To reliably evaluate the performance of SOP-MS, label-free MS was used for proteomic analysis of one cell at a time and protein identification is solely based on the actual MS/MS spectra from the analyzed cell, which is the cornerstone of MS-based proteomics. Furthermore, once it works for label-free MS analysis, SOP-MS can be widely used for other types of MS analysis of single cells. A commonly accessible Q Exactive Plus MS platform was used for the development of SOP-MS and its application demonstration.
Evaluation of SOP-MS Performance Using Peptides and Low-Input Human Cell Lysates
To achieve precise proteome quantification of single cells the inventors systematically evaluated sample recovery and processing reproducibility using more uniform low-input (small) samples (i.e., cell lysates or protein digests) with and without DDM in single PCR tubes. Selected reaction monitoring (SRM)-based targeted proteomics was used to optimize DDM concentrations from 0.005% to 0.1% due to its demonstrated higher reproducibility and quantitation accuracy when compared to global proteomics. Heavy isotope-labeled EGFR pathway peptide standards at a fixed concentration were measured at different DDM concentrations. The best SRM signals for most EGFR pathway peptides was achieved with 0.01-0.02% DDM, where higher DDM concentration can saturate the LC column and thus greatly degrade chromatographic performance. For simple peptide standard mixtures, 0.015% DDM was demonstrated for enabling to increase SRM signals by 3-35-fold with an average of ˜20-fold improvement (
The inventors next evaluated the performance of SOP-MS by serial dilution of uniform human breast cancer MCF7 cell lysates at 0.05-2.5 ng (close to 0.5-25 cells in protein mass) in the low-bind 96-well PCR plate (Methods). For 0, 0.05, 0.25, 0.5 and 2.5 ng of proteins, after trypsin digestion the average number of identified peptides (protein groups) was 38(7), 47 (31), 214 (116), 639 (293) and 3971 (1241), respectively. With the use of a MaxQuant MBR (match-between-run) function, the number of identified peptides (protein groups) consequently increased to 110 (33), 217 (156), 928 (437), 1897 (717) and 5792 (1539), respectively (
SOP-MS for Label-Free Proteomic Analysis of Small Tissue Sections
With its demonstrated improvement in analyzing low-input samples, the inventors next evaluated whether SOP-MS can be used for label-free, global proteomics analysis of small numbers of cells derived from mouse uterine tissues (
To evaluate whether the identified proteins can be used to specify tissue regions, the inventors performed principal component analysis (PCA). The luminal epithelium and stroma regions were clearly segregated based on the protein expression alone with the three biological replicates from the same regions being clustered together (
SOP-MS for Label-Free Quantitative Single-Cell Proteomics
With the demonstrated performance for small numbers of cells, the inventors evaluated whether SOP-MS can be used for proteomic analysis of single mammalian cells. Single cells were sorted directly into single low-bind PCR tubes (one cell per tube) by fluorescence-activated cell sorting (FACS). Single MCF10A cells were processed without and with 0.015% DDM (three biological replicates per condition) in parallel by SOP followed by LC-MS analysis (
To increase the number of identified unique peptides (protein groups), other commonly used proteomic algorithms were used to reanalyze the single-cell data. With the use of MBR function in MaxQuant, the average protein identifications were increased to 229, and a total of 384 protein groups were identified across three biological replicates for single MCF10A cells (
To validate SOP-MS for single-cell proteomics analysis the inventors performed an independent experiment for 4 single cells sorted by FACS from newly cultured MCF10A cells. An average of 146 protein groups were identified with the MS/MS spectra (
Application of SOP-MS to Single Cells Derived from a PCDX Model
To demonstrate the potential applications of SOP-MS to cancer research as well as to evaluate whether identification of hundreds of relatively abundant proteins can provide meaningful biological insights into cellular heterogeneity, the inventors applied SOP-MS for single-cell proteomic analysis of primary tumors and early lung metastases in a PCDX mouse model generated from patient CTCs (
Unsupervised PCA analysis has shown distinct clustering of proteins from the primary CTCs versus the lung metastases (
To further validate label-free MS quantification, two representative proteins, VIM and S100A9, were selected with median expression upregulated and downregulated by 4.7 and 8.6 in the lung metastatic cells, respectively (
Discussion
SOP-MS is a convenient robust method for label-free single-cell proteomics, where single cells are processed in either low-bind single tubes or multi-well plates which are routinely used for single-cell genomics and transcriptomics. The performance of SOP-MS (e.g., sensitivity, reproducibility, and quantitation accuracy) was demonstrated by label-free MS analysis of low mass inputs from serial dilution of uniform MCF7 cell lysates, LCM-dissected small tissue sections, and FACS-sorted single cells. Based on the actual MS/MS spectra for reliable protein identification (without using the MBR function) which is the cornerstone of MS-based proteomics, SOP-MS can identify ˜146 protein groups from single human cells, higher than ˜128 for iPAD1-M524 and 51 for OAD-M525 and ˜1.4-2.5-fold lower than ˜211-362 for nanoPOTS-M555-57 (Table 1), and ˜1200 proteins from small tissue sections (close to ˜20 cells). Comparative analysis of single MCF10A cells using both SOP-MS and nanoPOTS-MS has shown that the number of protein groups from SOP-MS is ˜1.6-fold lower than that from nanoPOTS-MS and ˜60% of protein groups from SOP-MS overlapped with the protein groups from nanoPOTS-MS (
With its demonstration for label-free MS analysis, SOP-MS can be equally used for other types of single-cell proteomic analysis (e.g., targeted proteomics and TMT-based MS analysis). It can also be used for analysis of other ultrasmall precious clinical specimens (e.g., rare CTCs and tissues from fine needle aspiration biopsy). The inventors have initially evaluated integration of our recently developed TMT-based BASIL strategy58 into SOP-MS for multiplexed analysis of 9 single MCF10A cells. A median correlation coefficient of ˜0.95 was achieved (
Future developments will focus on improvements in detection sensitivity and sample throughput for rapid deep proteome profiling of single mammalian cells. Enhancing detection sensitivity could be achieved by effective integration of ultralow-flow LC or capillary electrophoresis (CE) and a high-efficiency ion source/ion transmission interface with the most advanced MS platform. Further improvement can be gained by further reducing sample loss (e.g., systematic evaluation of different types of MS-friendly surfactants) and increasing reaction kinetics through reducing processing volume from 10-15 μL down to 1-2 μL with automated small-volume liquid handling (e.g., automated MANTIS liquid handler). All these improvements in detection sensitivity will lead to greatly increase the measurement reliability (e.g., more high-quality MS/MS spectra) as well as the number of identified peptides/protein groups. Sample throughput could be increased by using ultrafast high-resolution ion mobility-based gas-phase separation (e.g., SLIM59) to replace current slow liquid-phase (LC or CE) separation, and effective integration of liquid- and gas-phase separations (e.g., SLIM59 or FAIMS60) for greatly reducing separation time but without trading off separation resolution. Alternatively, sample multiplexing with isobaric barcoding and implementation of a multiple LC column system can also be considered to increase sample throughput. All these improvements could lead to a more powerful SOP-MS platform and will certainly close the gap between single-cell proteomics and single-cell transcriptomics or genomics.
When compared to proteomic analysis of bulk cells that only provides the averaged expression signal, single-cell proteomics can provide a clean signal for single cells of interest without signal contribution from other types of cells, allowing to uncover new biological discoveries. When applied for analysis of single cells derived from a clinically relevant PCDX model, SOP-MS can reveal distinct protein signatures between primary and metastatic tumors as well as cellular heterogeneity within the same cell type. Proteins with altered expression levels are involved in tumor immunity (e.g., S100A family members61), epithelial cell differentiation (e.g., CDSN), and EMT (vimentin38, 62), suggesting possible selective pressure for immune evasion and cell state plasticity. The data provide a clear path for future mechanistic studies of cancer metastasis with the potential to guide targeted cancer therapy. SOP-MS analysis of single cells is under way to reveal robust protein signatures related to physiological and pathological states at the single-cell resolution. Furthermore, with its demonstration for analysis of CTC-derived single cells, SOP-MS can be equally applied to clinically important patient CTCs that link disseminated and primary tumors. Thus, it has great potential for liquid biopsy-guided diagnostic and prognostic applications as well as for rational therapeutic intervention.
In summary, the inventors report an easily implementable SOP-MS method that capitalizes on using surfactant-assisted one-pot sample preparation to reduce the surface adsorption losses for label-free single-cell proteomics. Label-free quantitative proteome profiling of single cells can be achieved with easily accessible sample preparation devices (single tubes or multi-well plates) and standard LC-MS platforms. With its convenient features, SOP-MS can be readily implemented in any MS laboratory for single-cell proteomic analysis. The application of SOP-MS to single cells derived from a PCDX model demonstrated its power for precise characterization of cellular heterogeneity and discovery of distinct protein signatures related to breast cancer metastasis. With improvements in detection sensitivity and sample throughput as well as automation for high sample throughput, the inventors believe that SOP-MS has great potential to close the gap between single-cell proteomics and single-cell transcriptomics, and could open an avenue for single-cell proteomics with broad applicability in the biological and biomedical research.
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Methods
Human Sample Collection and Animal Studies
The human blood analyses for breast cancer patients were approved by the Institutional Review Boards at Northwestern University and complied with NIH guidelines for human subject studies. Animal procedures and experimental procedures have been performed under approval by Northwestern University Animal Care and Use Committee (ACUC) and complied with the NIH Guidelines for the Care and Use of Laboratory Animals. 8-10 weeks old female NSG mice were used for implantation of human breast cancer PCDX models and kept in specific pathogen-free facilities in the Animal Resources Center at Northwestern University. Breast tumors were harvested after 2-3 months and confirmed as a human PCDX with positive expression of human epithelial markers EpCAM, HER2, and CD44 as well as negative expression of mouse H-2Kd.
Reagents
n-Dodecyl β-D-maltoside (DDM), dithiothreitol (DTT), iodoacetamide (IAA), ammonium bicarbonate, acetonitrile, and formic acid were obtained from Sigma-Aldrich (St. Louis, Mo.). Promega trypsin gold was purchased from Promega Corporation (Madison, Wis.). Synthetic heavy peptides labeled with 13C/15N on the C-terminal arginine or lysine were purchased from New England Peptide (Gardner, Mass.).
Cell Culture
The MCF10A (MCF7) breast cancer cell line was obtained from the American Type Culture Collection (Manassas, Va.) and was grown in culture media63. Briefly, MCF10A (MCF7) cells were cultured and maintained in 15 cm dishes in ATCC-formulated Eagle's minimum essential medium (Thermo Fisher Scientific) supplemented with 0.01 mg/mL human recombinant insulin and a final concentration of 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, Mass.) with 1% penicillin/streptomycin (Thermo Fisher Scientific). Cells were grown at 37° C. in 95% O2 and 5% CO2. Cells were seeded and grown until near confluence.
MCF7 Cell Lysates
MCF7 cells were rinsed twice with ice-cold phosphate-buffered saline (PBS) and harvested in 1 mL of ice-cold PBS containing 1% phosphatase inhibitor cocktail (Pierce, Rockford, Ill.) and 10 mM NaF (Sigma-Aldrich). Cells were centrifuged at 1500 rpm for 10 min at 4° C., and excess PBS was carefully aspirated from the cell pellet. Cell pellets were resuspended in ice-cold cell lysis buffer (250 mM HEPES, 8 M urea, 150 mM NaCl, 1% Triton X-100, pH 6.0) at a ratio of ˜3:1 lysis buffer to cell pellet. Cell lysates were centrifuged at 14,000 rpm at 4° C. for 10 min, and the soluble protein fraction was retained. Protein concentrations were determined by the BCA assay (Pierce).
Fluorescence-Assisted Cell Sorting (FACS) of Single Cells
Prior to cell collection, PCR tubes or 96-well PCR plates were pretreated with 0.1% DDM for coating the surface and later the DDM solution was removed. The pretreated PCR tubes or 96-well PCR plates were air-dried in the fume hood. To avoid cell clumping, after detaching they were dispersed into a single-cell suspension by passing three times through a 25-gauge needle. The cells were suspended in PBS, and pelleted by centrifuging 5 min at 500 g. This process was repeated five times to remove the remaining PBS and trypsin. After that the cells were resuspended in PBS and passed through a 35 μm mesh cap (BD Biosciences, Canaan, CT) to remove large aggregates. A BD Influx flow cytometer (BD Biosciences, San Jose, Calif.) was used to deposit cells into the precoated PCR tubes. Alignment into a Hard-Shell 96-well PCR plate (Bio-Rad, Hercules, Calif.) was done using fluorescent beads (Spherotech, Lake Forest, Ill.), after which the coated PCR tubes were placed into the plates for cell collection. For unstained MCF10A cells, forward and side scatter detectors were used for cell identification. Once sorting gates were established, cells were sorted into the PCR tubes using the 1-drop single sort mode. After isolation of the desired number of cells into the PCR tube, the isolated cells were immediately centrifuged at 1000 g for 10 min at 4° C. to keep the cells at the bottom of the tube to avoid potential cell loss. The PCR tubes with the isolated cells were stored in a −80° C. freezer until further analysis.
Laser Capture Microdissection (LCM) of Tissue Sections
Prior to LCM experiments, a cap of PCR tube was prepopulated with a 5 μL water droplet. Laser capture microdissection (LCM) was performed on a PALM MicroBeam system (Carl Zeiss MicroImaging, Munich, Germany). Voxelation of the tissue section was achieved by selecting the area on the tissue using PalmRobo software, followed by tissue cutting and catapulting. Mouse uterine tissues containing two distinct cell types (luminal epithelium and stroma) were cut at an energy level of 42 and with an iteration cycle of 2 to completely separate 100 μm×100 μm tissue voxels at a thickness of 10 μm. The “CenterRoboLPC” function with an energy level of delta 10 and a focus level of delta 5 was used to catapult tissue voxels into the cap. The “CapCheck” function was activated to confirm successful sample collection from tissue sections to water droplets. After tissue collection into the droplet of the cap, the PCR tube was immediately centrifuged at 1000 g for 10 min at 4° C. to keep collected tissues at the bottom of the tube to avoid potential sample loss. The collected samples were processed directly or stored at −80° C. until use.
PCDX Model Generation and Dissociation of PCDX Tumors and Lungs
The PCDX-205 model was created by implanting prospective CTCs upon lysis of red blood cells (lysis buffer Sigma cat #R7757) and depletion of CD45+ PBMCs (Miltenyi Biotec Depletion column cat #130-042-901) from the blood cells of a breast cancer patient (NU-205) into the mammary fat pads of NSG mice. Breast tumors were harvested after 2-3 months and confirmed as a human PCDX with positive expression of human epithelial markers EpCAM, HER2, and CD44 as well as negative expression of mouse H2Kd. Tumor cells were lentiviral labeled by L2T64 which was generated by using the Luc2 and td Tomato sequences with connection by the short linker, 5′-GGAGATCTAGGAGGTGGAGGTA-GCGGTGGAGGTGGAAGCCAGGATCC-3′ (SEQ ID NO: 8). The L2T gene sequence was removed from a pCDNA3.1+ vector and placed within the pFUG lentiviral vector using traditional blunt end cloning. The spontaneous lung metastases were detected by IVIS of the lungs when dissected from the mice.
L2T+ PCDX-205 primary tumors and the lungs were harvested and briefly washed in PBS. Tissue was transferred to a Petri dish containing 10 mL dissociation media (RPMI 1640 media with 20 mM HEPES buffer), then minced into fine pieces. 400 μL of Liberase TH enzyme (Roche cat #5401135001) and 100 Units of DNase enzyme (Sigma cat #D4263) were added to the dissociation media, and the Petri dishes containing the tissues were transferred to an incubator at 37° C. and 5% CO2 for 2 h to complete dissociation. Tissue suspension was mixed every 15 min using a 10 mL serological pipette to aid dissociation. After tissue was completely digested into single cells, the solution was transferred to a 50 mL conical tube. The original petri dish was washed with 15 mL RPMI media containing 2% fetal bovine serum (FBS) (Sigma) and 1% penicillin/streptomycin (Gibco) and the contents transferred to a 50 mL conical tube containing the tissue solution to stop the dissociation reaction. Samples were centrifuged at 300 g for 5 min at 4° C., and the supernatant was removed. Samples were resuspended in 4 mL Red Blood Cell Lysing Buffer (Sigma) and kept on ice for 10 min, after which 20 mL of HBSS (Corning) was added to samples and centrifuged at 300 g for 5 min at 4° C. and the supernatant was removed. Samples were resuspended in 20 mL HBSS and filtered with a 40 μm filter. Cell numbers were counted, and samples were stored on ice until ready for use.
Single Cell Sorting of Patient CTCs from PCDXs and Early Metastases to the Lungs
Cells from dissociated tumor and lung tissues were washed in PBS and then centrifuged at 300 g for 5 min at 4° C. Samples were resuspended in 2% FBS in PBS. MDA-MB-231 cells were collected and suspended in 2% FBS in PBS to serve as a tdTomato (L2T)-negative control for flow analysis. Cancer cells from the tumor and lung samples were sorted based on L2T expression. L2T+ tumor cells of the lung metastases were initially sorted into 10% FBS in PBS prior to single cell sorting, and each of the L2T+ single cells from the primary tumor and lung metastases was sorted into 5 μL H2O in a single tube of a 96-tube PCR plate. Plates were sealed, briefly spun on a microplate centrifuge, and stored at −80° C. until later SOP-MS analysis.
Immunohistochemistry Staining
Formalin-fixed and paraffin-embedded tissues were processed and sectioned according to routine protocols. Heat mediated antigen retrieval was used prior to all staining procedures. Tissues were incubated with vimentin antibody (1:200 dilution, clone D21H3, Cell Signaling Technology) or S100A9 antibody (1:100 dilution, provided by Dr. Philippe Tessier at Laval University) overnight at 4° C. Antigen was detected using the EnVision+ Dual Link System (Dako) and counterstained with hematoxylin. Images were taken using a Leica DM4000B microscope and a Leica MC120 HD camera with a 40× objective.
Cell Lysis, Reduction, Alkylation, and Trypsin Digestion
For FACS-isolated cells, 2 μL of 0.1% DDM in 25 mM ammonium bicarbonate (ABC) was added to the PCR tube or each well of the 96-well plate. Intact cells were sonicated at 1-min intervals for 5 times over ice for cell lysis and centrifuged for 3 min at 3000 g. 0.3 μL of 100 mM DTT in 25 mM ABC was added to the PCR tube. Samples were incubated at 75° C. for 1 h for denaturation and reduction. After that, 0.5 μL of 60 mM IAA in 25 mM ABC was added to the PCR tube. Samples were incubated in the dark at room temperature for 30 min for alkylation. The reduction and alkylation steps appear optional: there is no apparent difference in protein identification and quantification between samples with and without reduction and alkylation. 2 of 1 trypsin (Promega) in 25 mM ABC was added to the PCR tube or the 96-well plate at a total amount of 2 ng. Samples were digested for ˜3-4 h at 37° C. with gentle sharking at ˜500 g. After digestion, 0.5 μL of 5% FA was added to the tube to stop enzyme reaction. The final sample volume was adjusted to ˜10-15 μL with the addition of 25 mM ammonium bicarbonate (triethylammonium bicarbonate for TMT samples) for direct LC injection. The sample PCR tube was inserted into the LC vial or the 96-well PCR plate was sealed with a matt. They were either analyzed directly or stored at −20° C. for later LC-MS analysis. For the integrated SOP-BASIL-MS analysis, the digested peptides from single MCF10A cells were labeled with different TMT reagents as sample channels, and 10 ng of peptides from bulk MCF10A cell digests were labeled with TMT126 as the carrier channel. The TMT126 labeled carrier channel peptides were equally distributed to each sample channel, and all the samples were combined together to form one single sample. The combined channel sample was desalted by using a simple reversed phase-based Stage Tip65.
For LCM-dissected tissue sections, 1.5 μL of cell lysis buffer containing 0.2% DDM and 5 mM DTT was added to the PCR tube and incubated at 80° C. for 60 min for cell lysis and protein denaturation. IAA was added to the PCR tube with the final concentration of 10 mM. Samples were incubated in the dark at room temperature for 30 min. After that they were diluted by the addition of 25 mM ammonium bicarbonate to reduce the DDM concentration to 0.02%. The mixed Lys-C and trypsin were added to the PCR tube with the final enzyme concentration of 0.5 ng/μL (i.e., a total of 5 ng for the final processing volume of 15 μL). The sample was gently mixed at 850 rpm for 3 min, and then incubated at 37° C. overnight (˜16 h) for digestion. After digestion, 1 μL of 5% FA was added to the PCR tube to stop enzyme reaction. The sample PCR tube was inserted into the LC vial and the sample was either directly analyzed or stored at −20° C. for later LC-MS analysis.
LC-MS/MS Analysis
The single-cell digests were analyzed using a commonly available Q Exactive Plus Orbitrap MS (Thermo Scientific, San Jose, Calif.). The standard LC system consisted of a PAL autosampler (CTC ANALYTICS AG, Zwingen, Switzerland), two Cheminert six-port injection valves (Valco Instruments, Houston, USA), a binary nanoUPLC pump (Dionex UltiMate NCP-3200RS, Thermo Scientific), and an HPLC sample loading pump (1200 Series, Agilent, Santa Clara, USA). Both SPE precolumn (150 μm i.d., 4 cm length) and LC column (50 μm i.d., 70-cm Self-Pack PicoFrit column, New Objective, Woburn, USA) were slurry-packed with 3-μm C18 packing material (300-A pore size) (Phenomenex, Terrence, USA). Sample was fully injected into a 20 μL loop and loaded onto the SPE column using Buffer A (0.1% formic acid in water) at a flow rate of 5 μL/min for 20 min. The concentrated sample was separated at a flow rate of 150 nL/min and a 75 min gradient of 8-35% Buffer B (0.1% formic acid in acetonitrile). The LC column was washed using 80% Buffer B for 10 min and equilibrated using 2% Buffer B for 20 min. Q Exactive Plus Orbitrap MS (Thermo Scientific) was used to analyze the separated peptides. A 2.2 kV high voltage was applied at the ionization source to generate electrospray and ionize peptides. The ion transfer capillary was heated to 250° C. to desolvate droplets. The data dependent acquisition mode was employed to automatically trigger the precursor scan and the MS/MS scans. Precursors were scanned at a resolution of 35,000, an AGC target of 3×106, a maximum ion trap time of 50 ms (100 ms for CTC single cell analysis). Top-10 precursors were isolated with an isolation window of 2, an AGC target of 2×105, a maximum ion injection time of 300 ms (for CTC single-cell analysis, the AGC target of 2×105 and 500 ms ion injection time was used), and fragmented by high energy collision with an energy level of 32%. A dynamic exclusion of 30 s was used to minimize repeated sequencing. MS/MS spectra were scanned at a resolution of 17,500.
Data Analysis
The freely-available open-source MaxQuant software was used for protein identification and quantification. The MS raw files were processed with MaxQuant (Version 1.5.1.11)66, 67 and MS/MS spectra were searched by Andromeda search engine against the against a human (or mouse) UniProt database (fasta file dated Apr. 12, 2017) (with the following parameters: tryptic peptides with 0-2 missed cleavage sites; 10 ppm of parent ion tolerance; 0.6 Da of fragment ion mass tolerance; variable modifications (methionine oxidation). Search results were processed with MaxQuant and filtered with a false discovery rate ≤1%. When a peptide library was available, the match between runs (MBR) function was selected to increase proteome coverage. Protein quantification was performed by using the label-free quantitation (LFQ) function. Contaminants were removed from the peptides.txt file prior to use for downstream statistical analysis. Biological functions and signaling pathways were analyzed by using DAVID Bioinformatics Resources (Version 6.8)68 and Peruses (Version 1.6.2.1)69, and protein-protein association network analysis was performed by the latest version of STRING (Version 11.0)70.
Statistics and Reproducibility
At least three biological or technical replicates were used to evaluate reproducibility for sample recovery and SOP-MS. No data exclusion was performed, and no randomization or blinding methods were used in data analysis. After label-free quantification with MaxQuant MBR, the extracted ion chromatogram (XIC) areas of the identified protein groups were log 2 transformed, and then normalized by the median value of each column. The proteins containing at least 50% valid values in one group were kept in the data matrix, and the missing values were imputed by the normal distribution in each column with a width of 0.3 and a downshift of 1.8 by using Perseus (Version 1.6.2.1)69. The non-supervised PCA analysis was used to generate PCA plot. The inventors further used Anova t-test to prioritize significantly differentiated proteins (p<0.05, FDR<0.2) for the heatmap generation. The extracted data were further processed and visualized with Microsoft Excel 2017.
It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
Stable Isotope-Labeled Phosphopeptides.
Crude stable isotope-labeled (SIL) phosphopeptides were synthesized with 13C/15N on C-terminal lysine or arginine (New England Peptide, Gardner, Mass.). The peptides were dissolved individually in 15% acetonitrile (ACN) and 0.1% formic acid (FA) at a concentration of 1.5 mM and stored at −80° C. A mixture of these peptides was made with a final concentration of 10 pmol/μL for each peptide.
LC-SRM Analysis.
The SIL phosphopeptides were diluted by ddH2O into 250 fmol/μL and analyzed using an Altis triple quadruple mass spectrometer (Thermo Fisher Scientific) equipped with a nanoACQUITY UPLC system (Waters, Milford, Mass.) for generating the data of
The selection of surrogate peptides for epidermal growth factor receptor (EGFR) pathway proteins and the SRM assays were described previously1. High-purity light peptides (>95%) were used to calibrate crude heavy peptide concentrations. Crude heavy isotope-labeled EGFR pathway peptide standards at a total amount of 30 fmol for each peptide were used for evaluation of peptide recovery with and without DDM (Table 1). Samples were analyzed using a nanoACQUITY UPLC (Waters Corporation, Milford, Mass.) coupled to a TSQ Vantage triple quadrupole mass spectrometer (Thermo Scientific, San Jose, Calif.). The UPLC's nanoACQUITY UPLC BEH 1.7 μm C18 column (75 μm i.d.×20 cm) was connected to a chemically etched 20 μm i.d. fused-silica electrospray emitter via a stainless metal union. Solvents used were 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in 90% acetonitrile (mobile phase B). An amount of ˜12 μL out of the total ˜15 μL peptide sample was directly loaded onto the BEH C18 column from the PCR tube without using a trapping column. Sample loading and separation were performed at a flow rate of 350 and 300 nL/min, respectively. The binary LC gradient was used: 5-20% B in 26 min, 20-25% B in 10 min, 25-40% B in 8 min, 40-95% B in 1 min and at 95% B for 7 min for a total of 52 min, and the analytical column was re-equilibrated at 99.5% A for 8 min. The TSQ Vantage mass spectrometer was operated with ion spray voltages of 2400±100 V, a capillary offset voltage of 35 V, a skimmer offset voltage of −5 V, and a capillary inlet temperature of 220° C. The tube lens voltages were obtained from automatic tuning and calibration without further optimization. The retention time scheduled SRM mode was applied for SRM data collection with the scan window of ≥6 min. The cycle time was set to 1 s, and the dwell time for each transition was automatically adjusted depending on the number of transitions scanned at different retention time windows. A minimal dwell time 10 ms was used for each SRM transition. All the EGFR pathway proteins were simultaneously monitored in a single LC-SRM analysis.
Data analysis. Skyline software was used for all SRM data analysis2. The raw data were initially imported into Skyline software for visualization of chromatograms of target peptides to determine the detectability of target peptides. For each peptide the best transition without matrix interference was used for precise quantification. Two criteria were used to determine the peak detection and integration: (1) same retention time and (2) approximately the same relative SRM peak intensity ratios across multiple transitions between endogenous (light) peptide and heavy peptide internal standards. All the data were manually inspected to ensure correct peak detection and accurate integration. The RAW data from TSQ Vantage were loaded into Skyline software to display graphs of extracted ion chromatograms (XICs) of multiple transitions of target proteins monitored.
Background of a PCDX Model.
In the dissemination of metastatic tumors, cancer cells from the primary tumor are shed into the peripheral blood vasculature. These circulating tumor cells (CTCs) serve as the vehicle by which primary tumors can seed distant metastases. In order to become a CTC, cancer cells from the primary tumor must undergo several steps to reach the bloodstream. Initially, tumor cells may undergo an epithelial to mesenchymal transition (EMT) and begin invading the surrounding extracellular matrix and basement membrane3-5. Eventually tumor cells will reach a local blood vessel and intravasate6. CTCs remain in the blood stream for up to several hours as single cells or clusters, sometimes associating with various other cell types, until they extravasate at a potential site of metastasis7-10. However, even in patients with advanced metastatic cancers, CTCs are a rare population (normally less than 0.1%) compared to peripheral blood mononuclear cells (PBMCs) within the blood. CTCs are commonly distinguished from other cell populations in the blood by negative expression of CD45, a leukocyte marker, and the positive expression of epithelial markers including EpCAM, cytokeratin, and/or other tumor associated antigens11, which might be heterogeneous and not expressed in all CTCs. There remains understudied concerning the dynamic changes CTCs may undergo compared to tumor cells within the primary tumor and distant metastases. Most notably, CTCs may exhibit cellular junction proteins and properties of cancer stem cells, which promote their ability to cluster and survive in the blood stream and seed distant metastases12-16. The detection of CTCs in singles and clusters in patient samples has shown important prognostic value7, 14-17. The characterization of CTC heterogeneity has been impeded due to the difficult sampling and maintenance of this rare population of tumor cells.
The development of patient derived xenografts (PDXs) that develop spontaneous metastases in mice has afforded researchers a representative model system to investigate the molecular and cellular basis of metastasis in vivo13, 18. In this study, the inventors further established patient CTC-derived xenografts (PCDXs) which developed spontaneous lung metastasis, first creation to our knowledge, for single cell proteomic profiling of primary tumor cells as well as spontaneous lung metastases. Lentiviral labeling of this PCDX with the luciferase 2-tdTomato (L2T) dual fusion gene reporter enabled a convenient isolation and FACS-based single cell sorting of L2T+ tumor cells from both primary tumor and lung metastasis after dissociation. The single cell proteomic profiling of PCDX model with metastasis not only allowed for the identification of new markers that can be leveraged for CTC isolation, but also facilitated elucidating the heterogeneous alterations of metastatic tumor cells upon colonization of the lungs.
Procedure for Prioritization of the 18 Differentially Expressed Proteins and Generation of the Heatmap:
1) After label-free quantification with MaxQuant MBR, the extracted ion chromatogram (XIC) areas of the identified protein groups were log 2 transformed, and then normalized by the median value of each column; 2) The proteins containing at least 50% valid values in one group were kept in the data matrix, and the missing values were imputed by the normal distribution in each column with a width of 0.3 and a downshift of 1.8 by using Perseus (Version 1.6.2.1); 3) The non-supervised PCA analysis was then used to generate PCA plot; 4) The inventors further used Anova t-test to prioritize significantly differentiated proteins between lung metastatic and primary tumor cells (p<0.05, FDR<0.2) for the heatmap generation.
Residual or minimal samples of small numbers of cells from PANC-1 and prostate cancer cell lines were prepared for mass spectrometry and treated with 0.015% DDM. Heavy isotope-labelled standards for peptides of interest were synthesized and used as standards. The inventors demonstrated that the disclosed methods are capable of detecting peptides derived from oncogenes, and single amino acid variants (SAAVs) of said peptides, e.g., SEQ ID NO: 1 (
C2660DTEID R
L330MVPFAMVIR
F310CTLVCPLHNQEVTAEDG
Y310CTLVCPLHNQEVTAEDG
L709AQMR
Q88LCDLR
S1025LALDK
S249PILTIITLEDSS
Y2660DTEIDR
W330MVPFAMVIR
T1025LALDK
LMVPFAMV1R
WMVPFAMVIR
C2660DTEIDR
L330MVPFAMVIR
Y123CK
D717TELR
L777GSPYVS
Q88LCDLR
K453 (SEQ
Q453DLLN
G453DLLN
E914DR
S1025LALDK
I155R
P156VR
H175CPHHER
L193LIR
R214
D244GMNR
S249PILTIIT
Citations to a number of patent and non-patent references may be made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.
This application claims benefit of priority to U.S. Provisional Application No. 63/151,537, filed Feb. 19, 2021, the content of which is incorporated herein by reference in its entirety.
This invention was made with government support under UG3CA256967 and CA223715 awarded by the National Institutes of Health and W81XWH-16-1-0021 awarded by the Department of Defense Breast Cancer Research Program (DOD BCRP). The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63151537 | Feb 2021 | US |
