Patent Application
20080038194
The present invention is based on a novel approach to the study of molecular biology, by inducing subtle modifications in the isotope ratio of particular peptides and polypeptides for expression proteomics. “Subtle,” as used herein with reference to the modification of isotopes included in target molecules, is defined as a “swapping” of, on average, an amount of isotopes included in target molecules such that there is a measurable effect upon the observed peptide isotope distribution, without causing a gross extension or displacement of the single isotope envelope. The modification introduced is gross compared to natural isotopic variability yet subtle compared with strategies that seek full exchange. Isotope ratio is calculated for specific peptides or polypeptides based upon their isotopic distributions obtained by high-resolution mass spectrometry. This requires either an estimate of elemental composition based upon mass and average amino acid elemental composition (averagine), or the precise elemental composition based upon the peptide sequence determined by tandem mass spectrometry and protein identification algorithms, as in typical proteomics experiments. Successful implementation of the present invention was demonstrated by modest elevation of the 13C/12C ratio in Synechocystis sp. PCC 6803 cultures as a model for other organisms. Small fluctuations of isotope ratios occur in living organisms as a result of metabolic bias and nutrition (Meier-Augenstein et al., “Applied gas chromatography coupled to isotope ratio mass spectrometry,” Journal of Chromatography, Vol. 842, pp. 351-371 (1999)). In this model, carbon was supplied as CO2/HCO3 (from bicarbonate) and entered the metabolic cycle via carbon fixation.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), provides one skilled in the art with a general guide to many of the terms used in the present application.
One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. Alternate methodologies and procedures may be readily implemented without undue experimentation. The presently disclosed embodiments are therefore to be considered in all respects as illustrative and not restrictive. Example methodologies and procedures may be readily understood by reference to the Results and Examples described below. Furthermore, all publications referred to herein are incorporated in their entirety.
Subtle modifications may be induced with reference to any suitable target isotope or isotopes, which may include, but are in no way limited to, 13C for 12C, 18O for 16O, and 15N for 14N. Additional isotopes suitable for subtle modification in accordance with alternate embodiments for the present invention will be readily appreciated by those of skill in the art; for instance, deuterium may be swapped for hydrogen.
Subtle alteration of the ratio of 13C to 12C may be particularly advantageous in connection with the methods of the present invention, insofar as these methods are implemented in connection with proteomics. Carbon is the most abundant constituent of proteins, and, thus, a small change in the ratio of 13C to 12C has the most dramatic effect upon isotopic distribution; changing this ratio from 100:1 to 100:2 (or 200:1), for instance, may have quite a dramatic effect. In the context of proteomics, this subtle alteration of isotopic ratio can be measured from the isotopic distribution of peptide ions; thereby providing a means of stable isotope tagging that does not require full conversion to a non-natural isotope. For example,
The methods of the present invention are by no means limited to the study of proteomics, however. In fact, the invention may find application in a host of biological systems, as well as non-biological systems (i.e., in the study of any system in which isotopes may be subtly modified and thereafter analyzed by the methods described herein).
Methods for inducing the subtle modifications incorporated in various aspects of the present invention will similarly be recognized by and may be readily implemented by those of skill in the art without undue experimentation. In addition to the use of variants of the technology described above (i.e., ICAT, SILAC, enzymatic exchange, and growth in stable isotopes), other means for inducing the subtle modification of isotopes can be readily ascertained. For example, a living animal may be fed a diet that includes the non-natural isotope or isotopes sought to be introduced into their internal physiology; diet influences actual isotope ratios in animals, including humans. Such a diet may include, for example, food (e.g., animal chow) supplemented with the non-natural isotope or, in another embodiment of the invention, the diet may include deuterated or deuterium-enriched water. Alternatively, the isotope may be introduced by pharmacological means (e.g., a diet supplement), or by other conventional forms of administration (e.g., injection of saline consisting of the non-natural isotope). The particular mode of adminstration may be selected based upon the physiological process to be studied.
Once the target molecule (or molecules) has been subtly modified, it may be studied by a number of different technologies. For example, isotope ratio mass spectrometry (“IRMS”) may be employed, whereby levels of different isotopes (e.g., 13C and 12C) may be measured after conversion to carbon dioxide (e.g., by combustion). Alternatively, the isotope ratio may be measured by calculation from the peptide mass spectra obtained by various forms of mass spectrometry (e.g., MSMS). Specific protein molecules can be identified by MSMS in connection with an appropriate database search, or isotopic distribution can be estimated using averagine (i.e., a model amino acid with elemental components occurring at frequencies deduced from the PIR database). In particular embodiments of the invention, high resolution mass spectrometry, such as Fourier transform mass spectrometry (“FTMS”), may be particularly advantageous and accurate. Moreover, in instances where peptide sequences (and, thus, elemental compositions) are known, the measurements attainable on high-resolution instrumentation, such as mass spectrometers employing FTMS, may be highly accurate.
The present invention has a range of applications. In one embodiment, the invention may be used in connection with isotope coding by subtle alteration of isotope ratio in proteins, peptides and polypeptides. This may be particularly useful in connection with proteomics. For instance, one may study the relative expression of various proteins in a biological system. Two (or more) samples can be distinguished by their isotope ratios; thereby allowing mixing and relative expression measurement by comparison of peak height/areas (i.e., in a MALDI readout). The isotope ratio of a peptide in a mixture is determined by relative contribution from non-labeled as compared with labeled material.
In another embodiment, the present invention may be used to study protein turnover (e.g., one may monitor metabolic or transcriptional activity by seeking out newly altered or transcribed proteins, respectively). Protein turnover may be measured using pulse-chase protocols. For instance, if a human begins to eat food with an altered isotope ratio, then this will first be observed in rapidly turning-over proteins. Slow-turning proteins will be last to manifest the change in isotope ratio.
Among the benefits of employing the methods of the present invention in connection with, for example, proteomics, is the significant cost savings and the ability to study systems that were heretofore impossible to study by way of isotope swapping.
The 13C/12C isotope ratio of Synechocystis sp. PCC 6803 grown in culture was altered modestly via manipulation of the source of CO2 for photoautotrophic growth (bicarbonate). Soluble proteins were separated from the membrane fraction after mechanical disruption of the cells. Membrane proteins were precipitated with acetone, dissolved in formic acid and subjected to analysis by liquid chromatography electrospray-ionization mass spectrometry with fraction collection (LC-MS+). Fractions were reduced, alkylated and digested with trypsin prior to MALDI-TOF analysis.
prob(n)=combin(X,n)*Pn*(1−P)(X−n).
Given an elemental composition, Isosolv is used to estimate 13C/12C ratio. For any particular molecular weight, the number of carbons is estimated by dividing the molecular weight by 110 (the average mass of an amino acid) and multiplying by 4.94 (the average number of carbons per amino acid). In turn, given a measured isotopic distribution, the 13C probability can be determined by calculating the difference between the measured distribution and the theoretical distribution for an arbitrary 13C abundance. The estimated 13C abundance parameters can then be incrementally altered until the error between the theoretical distribution and the calculated distribution has been minimized, thereby yielding the 13C probability in the measured spectrum.
Table 1 summarizes the 13C/12C isotope ratios of peptides derived from their isotopic distributiona.
aThe Isosolv algorithm (experimental) was used to calculate 13C/12C ratio for the peptide FLSSTELQIAFGR (amino acids 18-30 phycocyanin A; Q54715; elemental composition C67 H106 N17 O20; calculated M + H+ 1468.7794 Da.
bTargeted 13C addition.
cSum of 300-500 laser flashes.
dLCQ-DECA single zoom scan data.
eUse of peak height versus area for calculation of isotope ratio.
fNot available.
There was a general decrease in signal to noise in the higher supplementation samples (see
It was desirable that the isotope coding strategy be compatible with existing protein identification protocols, thus, to that end, the protein identification performance was examined in the LC-MSMS experiments used for
Table 2 summarizes performance of the experiment when interrogated with Sequest for protein identifications. Protein identification performance was observed under altered 13C/12C ratiosa.
13C
aProtein identification used Sequest (ThermoFinnigan) to match experimental tandem mass spectrometry data to a database of translated Synechocystis sp. PCC 6803 open reading frames. ‘No enzyme’ is selected so that all possible sequences are screened. The results of a representative experiment are shown.
bThe cross-correlation coefficient (Xcorr) provides a measure of how well a tandem mass spectrum matches that predicted for a particular peptide. Searches that yield tryptic peptide matches with Xcorr > 2.3 are generally significant matches. Searches that yield tryptic peptide matches with Xcorr > 4.0 are nearly always highly significant matches with good signal to noise.
cThe delta correlation (ΔCn) is a measure of how well a number of peptide matches identify a specific protein.
It is noted that the lower 13C supplemented samples were unaffected with respect to peptides/proteins identified while a noticeable decline was observed for the highest 13C sample. The widening of the isotopic envelope may lead to less frequent triggering of the MSMS experiment as maximum signal intensities drop. Also MSMS spectra may fail to yield Sequest hits as higher mass b and y fragments become too large to fall within tolerance limits for database matching. In
Modification of 13C/12C was chosen because carbon is the most abundant element in peptides and proteins, though the strategy could also employ 15N:14N or 18O:16O manipulation as discussed above. Cargile (Cargile et al., “Synthesis/degradation ratio mass spectrometry for measuring relative dynamic protein turnover,” Analytical Chemistry, Vol. 76, pp. 86-97 (2004)) used pulse labeling with 13C to measure protein turnover kinetics although the use of high atom percentages of 13C lead to dramatically extended isotope distributions that in the proteomics context would result in dramatic loss of separation space and, as is apparent in the Figures presented, the appearance of peaks at every unit across the mass spectrum. The data presented in
For SMIRP to be useful in the context of expression proteomics it is necessary to control a number of variables such that significant changes in relative expression can be measured with quantifiable error. As the results show, there is significant variability associated with using relative isotopomer abundance for calculation of isotope ratio. While not wishing to be bound by any particular theory, it is believed that the origin of this variability is due to variability of relative isotopomer measurement by the mass spectrometer. Variability among different peptides as a result of their specific elemental compositions combined with metabolic bias, and natural variability of specific peptide isotope ratio based upon changes in flux that alter metabolic bias might also be significant in the context of proteomics. Measurement variability can be addressed by increased sampling and the use of mass spectrometers with improved sensitivity and resolution while the later natural variability must be explored in detail in future research. Thus, natural variability may provide additional information with respect to cellular flux in the future. SMIRP technology can be applied to any conceivable proteomics experiment including 2D gels, MuDPIT (Washburn et al., “Large-scale analysis of the yeast proteome by multidimensional protein identification technology,” Nature Biotechnology, Vol. 19, pp. 242-247 (2001)), accurate mass and time tags (Strittmatter et al, “Proteome analyses using accurate mass and elution time peptide tags with capillary LC time-of-flight mass spectrometry,” Journal of the American Society for Mass Spectrometry, Vol. 14, pp. 980-991 (2003)) and SILAC (Ong et al., “Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics,” Molecular and Cellular Proteomics, Vol. 1, pp. 376-386 (2002)).
The following examples illustrate a method of performing subtle isotope modification and monitoring peptide turnover. Modifications of these examples will be readily apparent to those skilled in the art seeking to perform isotope modification for expression proteomics or other applications differing from those described herein. These examples are included merely for purposes of illustration.
Synechocystis sp. PCC 6803 cells were grown autotrophically in liquid BG-11 medium (available from Sigma; St. Louis, Mo.) (Rippka et al., “Generic assignments, strain histories and properties of pure cultures of cyanobacteria,” Journal of General Microbiology, Vol. 111, pp. 1-61 (1979)) buffered with 5 mM N-tris-hydroxymethyl-2-aminoethanesulfonic acid (TES)-NaOH (pH 8.0) (available from Sigma) and supplemented with filter-sterilized mixture of NaH13CO2 (obtained from Cambridge Isotope Laboratories; 99% 13C; Andover, Mass.) and NaH12CO2 (obtained from Sigma, 1.1% of 13C). The bicarbonate mixture was added to the freshly autoclaved BG-11 medium, which contained essentially no dissolved CO2, and the bottles with the medium were sealed until further usage. The added NaH13CO3 was calculated to be 0%, 1.5%, 3.0%, or 6.0% of the total NaHCO3 taking into account natural 13C abundance (˜1 13C/100 12C) of the standard BG-11 medium (20 mg/l). Cultures were started by inoculating 50 ml of the medium (in a 150 ml flask) with a small amount of cells followed by incubation at 28° C. with light intensity of 50 mol photons/m2 s while shaking at 100 rpm on a rotary shaker. Every 2-3 days the cultures were transferred to larger flasks and diluted with fresh BG-11 medium containing an appropriate amount of NaH13CO3. The cultivation continued until the cell cultures reached OD730=0.5 in a total volume of 500 ml. After that, cells were harvested by centrifugation, washed with thylakoid buffer (50 mM MES-NaOH at pH 7.0, 5 mM CaCl2, 5 mM MgCl2, 10 mM NaCl, 15% v/v glycerol, and 0.5% v/v DMSO), and then frozen in liquid nitrogen.
Cells were thawed rapidly and placed on ice. Protease inhibitors (obtained from Sigma; P8465; 50 1/1 ml aliquot) were added prior to transfer of the cell suspension to tubes containing glass beads (0.1 mm; 1.0-1.2 g) pre-cooled on ice. Cells were broken using a micro-beadbeater (obtained from Biospec Products; Bartlesville, Okla.) on its maximum setting (4-5×30 s). Cells were cooled on ice between each treatment. Cell breakage efficiency was assessed by extraction of cells in acetone (10 μl cells plus 1 ml 80% acetone), agitation and centrifugation; chlorophyll was only extracted after cell breakage yielding a blue pellet. The broken cell suspension was diluted 10-fold with ice cold thylakoid buffer containing protease inhibitors and decanted to pre-cooled centrifuge tubes. Unbroken cells were removed (500 rpm SS34; 1 min) prior to transfer to clean tubes and sedimentation of the membranes (20,000 rpm SS34; 30 min). The supernatant was retained for soluble proteins and the pellet was resuspended in thylakoid buffer, homogenized (Teflon/glass) and stored at −80° C.
Samples of Synechocystis membranes were analyzed by LCMS+ (Whitelegge et al., 2002). Membrane fraction proteins (300-600 g) were precipitated at the interface of an aqueous chloroform/methanol phase separation (Wessel and Flugge, “A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids.,” Analytical Biochemistry, Vol. 138, pp. 141-43 (1984)) as described (Whitelegge et al., “Toward the bilayer proteome, electrospray ionization-mass spectrometry of large, intact transmembrane proteins,” Proceedings of the National Academy of Sciences of the United States of America, Session 96, pp. 10695-0698 (1999)). Precipitated proteins were recovered after removal of the aqueous phase and addition of methanol. Precipitated samples were dried at atmospheric pressure for 2 min (25° C.) and dissolved in 90% formic acid (available from Sigma; 100 μl) immediately prior to HPLC. Reverse phase chromatography (RPC) of intact proteins was performed as described previously (Whitelegge et al., 2002; Whitelegge, “Thylakoid membrane proteomics,” Photosynthesis Research, Vol. 78, pp. 265-277 (2003); and Whitelegge, “HPLC and mass spectrometry of intrinsic membrane proteins,” Methods in Molecular Biology, Vol. 251, pp. 323-340 (2004)) using a macroporous polymeric support (obtained Polymer Labs; Amherst, Mass.; PLRP/S, 300 A, 5 m, 2×150 mm) at 100 μl/min (40° C.). The column was previously equilibrated in 95% A, 5% B (A, 0.1% TFA in water; B, 0.05% TFA in acetonitrile/isopropanol, 1:1, v/v) and eluted with a compound linear gradient from 5% B at 5 min after injection, through 40% B at 30 minutes and to 100% B at 150 min. The eluent was passed through a UV detector (280 nm) prior to a liquid-flow splitter (inserted between HPLC detector and mass spectrometer) that made it possible to collect fractions concomitant with electrospray-ionization mass spectrometry (ESI-MS). Fused silica capillary was used to transfer liquid to the ESI source (˜50 cm) or fraction collector (˜25 cm). The split fractions were collected into micro-centrifuge tubes at 1 min intervals. ESI-MS was performed as described (Whitelegge et al., “Electrosprayionization mass spectrometry of intact intrinsic membrane proteins,” Protein Science, Vol. 7, pp. 1423-430 (1998)) using a triple quadruple instrument (obtained from Applied Biosystems; Foster City, Calif.; API III). Orifice voltage was ramped from 60 to 120 over the mass range acquired (600-2300) and the instrument scanned with a step size of 0.3 amu and 1 ms dwell. Data were processed using MacSpec 3.3, Hypermass or BioMultiview 1.3.1 software (obtained from Applied Biosystems).
Selected fractions collected during LCMS+ were reduced, alkylated and treated with trypsin (obtained from Promega; Madison, Wis.; sequencing grade modified by reductive methylation). DTT (15 μl, 10 mM in 50 mM ammonium bicarbonate; 30 min, 24° C.) then iodoacetamide (15 μl, 55 mM in 50 mM ammonium bicarbonate; 20 min, 24° C.) and finally trypsin (12.5 μl, 6 ng/l in 50 mM ammonium bicarbonate; 3 h, 37° C.) was added to aliquots of fractions (10 μl). After incubation, samples were dried by centrifugal evaporation and stored at −20° C. prior to analysis by LC-MSMS.
Dried reaction mixtures were re-dissolved in 5 μl of 70% acetic acid and analyzed (0.2 μl plus 0.5 μl matrix) by matrix-assisted laser desorption ionization (MALDI) coupled to delayed extraction time-of-flight MS in the reflector mode (obtained from Applied Biosystems; Voyager DE STR) using -cyano-4-hydroxycinnamic acid as matrix (10 mg/ml solution in water/acetonitrile/TFA 30/70/0.1) and internal/external calibration with bovine insulin. Manufacturer supplied default settings optimized for peptides less than 6000 Da were used for all samples.
Samples were analyzed by LC-MSMS with data-dependent acquisition (obtained from ThermoFinnigan; San Jose, Calif.; LCQ-DECA) after dissolution in 5 μl of 70% acetic acid (v/v). A reverse phase column (obtained from Michrom Biosciences, San Jose, CA; 200 m×10 cm; PLRP/S 5 m, 300 Å) was equilibrated for 10 min at 1.5 μl/min with 95% A, 5% B (A, 0.1% formic acid in water; B, 0.1% formic acid in acetonitrile) prior to sample injection. A linear gradient was initiated 10 min after sample injection ramping to 60% A, 40% B after 50 min and 20% A, 80% B after 65 min. Column eluent was directed to a coated glass electrospray emitter (TaperTip, TT150-50-50-CE-5, New Objective) at 3.3 kV for ionization without nebulizer gas. The mass spectrometer was operated in ‘triple-play’ mode with a survey scan (400-500 m/z), data-dependent zoom scan and MSMS. Individual sequencing experiments were matched to a custom Synechocystis sequence database using Sequest software (obtained from ThermoFinnigan).
For a carbon isotope distribution the probability of having ‘n’ 13C's given ‘X’ total carbons and a 13C probability of ‘P’ is described as follows:
prob(n)=combin(X,n)*Pn*(1−P)(X−n)
When given an elemental composition Isosolv uses this for estimation of 13C/12C ratio. When given a molecular weight, the number of carbons is estimated by dividing the molecular weight by 110 (the average mass of an amino acid) and multiplying by 4.94 (the average number of carbons per amino acid). Then, given a measured isotopic distribution, the 13C probability can be determined by calculating the difference between the measured distribution and the theoretical distribution for an arbitrary 13C abundance. The estimated 13C abundance parameters are then incrementally altered until the error between the theoretical distribution and the calculated distribution has been minimized thus yielding the 13C probability in the measured spectrum. The version of Isosolv used in these examples includes natural minor contributions of D, 15N, 17/18O only.
While the description above refers to particular embodiments of the present invention, it will be understood that many modifications may be made without departing from the spirit thereof. The accompanying claims are intended to cover such modifications as would fall within the true scope and spirit of the present invention. The presently disclosed embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims, rather than the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US05/01393 | 1/13/2005 | WO | 00 | 7/2/2007 |
| Number | Date | Country | |
|---|---|---|---|
| 60536766 | Jan 2004 | US |
