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Relative Protein Quantification by Isobaric SILAC with Immonium Ion Splitting (ISIS)

2007; Elsevier BV; Volume: 7; Issue: 5 Linguagem: Inglês

10.1074/mcp.m700440-mcp200

1535-9484

Mara Colzani, Frédéric Schütz, Alexandra Potts, Patrice Waridel, Manfredo Quadroni,

Metabolomics and Mass Spectrometry Studies

Metabolic labeling techniques have recently become popular tools for the quantitative profiling of proteomes. Classical stable isotope labeling with amino acids in cell cultures (SILAC) uses pairs of heavy/light isotopic forms of amino acids to introduce predictable mass differences in protein samples to be compared. After proteolysis, pairs of cognate precursor peptides can be correlated, and their intensities can be used for mass spectrometry-based relative protein quantification. We present an alternative SILAC approach by which two cell cultures are grown in media containing isobaric forms of amino acids, labeled either with 13C on the carbonyl (C-1) carbon or 15N on backbone nitrogen. Labeled peptides from both samples have the same nominal mass and nearly identical MS/MS spectra but generate upon fragmentation distinct immonium ions separated by 1 amu. When labeled protein samples are mixed, the intensities of these immonium ions can be used for the relative quantification of the parent proteins. We validated the labeling of cellular proteins with valine, isoleucine, and leucine with coverage of 97% of all tryptic peptides. We improved the sensitivity for the detection of the quantification ions on a pulsing instrument by using a specific fast scan event. The analysis of a protein mixture with a known heavy/light ratio showed reliable quantification. Finally the application of the technique to the analysis of two melanoma cell lines yielded quantitative data consistent with those obtained by a classical two-dimensional DIGE analysis of the same samples. Our method combines the features of the SILAC technique with the advantages of isobaric labeling schemes like iTRAQ. We discuss advantages and disadvantages of isobaric SILAC with immonium ion splitting as well as possible ways to improve it. Metabolic labeling techniques have recently become popular tools for the quantitative profiling of proteomes. Classical stable isotope labeling with amino acids in cell cultures (SILAC) uses pairs of heavy/light isotopic forms of amino acids to introduce predictable mass differences in protein samples to be compared. After proteolysis, pairs of cognate precursor peptides can be correlated, and their intensities can be used for mass spectrometry-based relative protein quantification. We present an alternative SILAC approach by which two cell cultures are grown in media containing isobaric forms of amino acids, labeled either with 13C on the carbonyl (C-1) carbon or 15N on backbone nitrogen. Labeled peptides from both samples have the same nominal mass and nearly identical MS/MS spectra but generate upon fragmentation distinct immonium ions separated by 1 amu. When labeled protein samples are mixed, the intensities of these immonium ions can be used for the relative quantification of the parent proteins. We validated the labeling of cellular proteins with valine, isoleucine, and leucine with coverage of 97% of all tryptic peptides. We improved the sensitivity for the detection of the quantification ions on a pulsing instrument by using a specific fast scan event. The analysis of a protein mixture with a known heavy/light ratio showed reliable quantification. Finally the application of the technique to the analysis of two melanoma cell lines yielded quantitative data consistent with those obtained by a classical two-dimensional DIGE analysis of the same samples. Our method combines the features of the SILAC technique with the advantages of isobaric labeling schemes like iTRAQ. We discuss advantages and disadvantages of isobaric SILAC with immonium ion splitting as well as possible ways to improve it. Methods for quantitative proteomics based on stable isotope labeling have become in the last decade very powerful tools to investigate cellular processes (1Tao W.A. Aebersold R. Advances in quantitative proteomics via stable isotope tagging and mass spectrometry.Curr. Opin. Biotechnol. 2003; 14: 110-118Crossref PubMed Scopus (245) Google Scholar, 2Chen X. Sun L. Yu Y. Xue Y. Yang P. Amino acid-coded tagging approaches in quantitative proteomics.Expert Rev. Proteomics. 2007; 4: 25-37Crossref PubMed Scopus (77) Google Scholar, 3Ong S.E. Mann M. Mass spectrometry-based proteomics turns quantitative.Nat. Chem. Biol. 2005; 1: 252-262Crossref PubMed Scopus (1321) Google Scholar). The main applications of such methods have been the analysis of changes in protein expression (4Brand M. Ranish J.A. Kummer N.T. Hamilton J. Igarashi K. Francastel C. Chi T.H. Crabtree G.R. Aebersold R. Groudine M. Dynamic changes in transcription factor complexes during erythroid differentiation revealed by quantitative proteomics.Nat. Struct. Mol. Biol. 2004; 11: 73-80Crossref PubMed Scopus (190) Google Scholar) as well as the elucidation of networks of molecular (protein-protein (5Blagoev B. Kratchmarova I. Ong S.E. Nielsen M. Foster L.J. Mann M. A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling.Nat. Biotechnol. 2003; 21: 315-318Crossref PubMed Scopus (607) Google Scholar) or DNA-protein) interactions or the study of post-translational modifications (6Gruhler A. Olsen J.V. Mohammed S. Mortensen P. Faergeman N.J. Mann M. Jensen O.N. Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway.Mol. Cell. Proteomics. 2005; 4: 310-327Abstract Full Text Full Text PDF PubMed Scopus (698) Google Scholar, 7Sachon E. Mohammed S. Bache N. Jensen O.N. Phosphopeptide quantitation using amine-reactive isobaric tagging reagents and tandem mass spectrometry: application to proteins isolated by gel electrophoresis.Rapid Commun. Mass Spectrom. 2006; 20: 1127-1134Crossref PubMed Scopus (51) Google Scholar). Isotope labeling methods can be classified in two broad classes. Those based on residue-specific chemical derivatization with labeled reagents have the great advantage of offering greater flexibility in the choice of the chemistry and are universally applicable. However, they can suffer from the complexity of the steps involved and from the risk of side reactions. Metabolic labeling approaches, on the other hand, are only possible for organisms whose cells can be cultured in strictly controlled conditions. They offer, however, the advantage that no additional labeling steps are involved and that the proteins maintain all their native properties. Such techniques are especially attractive when complex biochemical purifications are necessary to obtain the samples to be studied because extracts of cells can be mixed at the very beginning of the procedure, thereby eliminating artifacts due to slight variations in the purification conditions for the different samples. Many chemical labeling schemes have been devised. In turn, they can be subdivided in two groups based on the approaches needed for quantification. Most techniques rely on measuring the intensity of light/heavy parent ions in MS survey scans to establish intensity ratios. One technical issue with such an approach is the need to establish an unambiguous correlation (based on m/z and retention times in liquid chromatography) between tandem MS spectra used for identification and their precursor peaks in MS survey scans. ICAT (8Gygi S.P. Rist B. Gerber S.A. Turecek F. Gelb M.H. Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.Nat. Biotechnol. 1999; 17: 994-999Crossref PubMed Scopus (4362) Google Scholar) and a multitude of other labeling schemes (9Goodlett D.R. Keller A. Watts J.D. Newitt R. Yi E.C. Purvine S. Eng J.K. von Haller P. Aebersold R. Kolker E. Differential stable isotope labelling of peptides for quantitation and de novo sequence derivation.Rapid Commun. Mass Spectrom. 2001; 15: 1214-1221Crossref PubMed Scopus (271) Google Scholar, 10Munchbach M. Quadroni M. Miotto G. James P. Quantitation and facilitated de novo sequencing of proteins by isotopic N-terminal labelling of peptides with a fragmentation-directing moiety.Anal. Chem. 2000; 72: 4047-4057Crossref PubMed Scopus (251) Google Scholar, 11Schmidt A. Kellermann J. Lottspeich F. A novel strategy for quantitative proteomics using isotope-coded protein labels.Proteomics. 2005; 5: 4-15Crossref PubMed Scopus (434) Google Scholar, 12Zhou H. Ranish J.A. Watts J.D. Aebersold R. Quantitative proteome analysis by solid-phase isotope tagging and mass spectrometry.Nat. Biotechnol. 2002; 20: 512-515Crossref PubMed Scopus (370) Google Scholar) belong to this class. The second group is formed by techniques that exploit quantitative information embedded in tandem MS spectra rather than in survey scans (13Thompson A. Schafer J. Kuhn K. Kienle S. Schwarz J. Schmidt G. Neumann T. Johnstone R. Mohammed A.K. Hamon C. Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS.Anal. Chem. 2003; 75: 1895-1904Crossref PubMed Scopus (1757) Google Scholar, 14Ross P.L. Huang Y.N. Marchese J.N. Williamson B. Parker K. Hattan S. Khainovski N. Pillai S. Dey S. Daniels S. Purkayastha S. Juhasz P. Martin S. Bartlet-Jones M. He F. Jacobson A. Pappin D.J. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.Mol. Cell. Proteomics. 2004; 3: 1154-1169Abstract Full Text Full Text PDF PubMed Scopus (3721) Google Scholar). Earlier examples of this principle used protease-mediated 16O/18O labeling (15Heller M. Mattou H. Menzel C. Yao X. Trypsin catalyzed 16O-to-18O exchange for comparative proteomics: tandem mass spectrometry comparison using MALDI-TOF, ESI-QTOF, and ESI-ion trap mass spectrometers.J. Am. Soc. Mass Spectrom. 2003; 14: 704-718Crossref PubMed Scopus (141) Google Scholar, 16Wang Y.K. Ma Z. Quinn D.F. Fu E.W. Inverse 18O labelling mass spectrometry for the rapid identification of marker/target proteins.Anal. Chem. 2001; 73: 3742-3750Crossref PubMed Scopus (98) Google Scholar, 17Schnolzer M. Jedrzejewski P. Lehmann W.D. Protease-catalyzed incorporation of 18O into peptide fragments and its application for protein sequencing by electrospray and matrix-assisted laser desorption/ionization mass spectrometry.Electrophoresis. 1996; 17: 945-953Crossref PubMed Google Scholar). Such approaches, however, require co-fragmentation of the precursors, and this necessitates very close or identical masses, such as in the iTRAQ 1The abbreviations used are: iTRAQ, isobaric tags for relative and absolute quantitation; SILAC, stable isotope labeling with amino acids in cell cultures; ISIS, immonium ion splitting; 2D, two-dimensional; 1D, one-dimensional; FDR, false discovery rate; H/L, heavy/light; QQ-TOF, quadrupole/quadrupole TOF. scheme. The latter is a special case that uses isobaric tagging reagents that fragment to give, for every peptide, diagnostic low mass ions used for quantification. Compared with methods with distinct mass precursors, these techniques present the advantages that no precursor mass splitting results in higher signal intensity and data handling is easier because only MS/MS data are needed, thus eliminating the need to integrate peaks along chromatographic runs. Appropriate design of the reagent has allowed the reporter ion mass to be varied, and this has been used to implement 4- and 8-fold multiplexing work flows. By contrast, classical SILAC (18Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4625) Google Scholar) is a metabolic labeling method similar, from the point of view of the quantification procedure, to non-isobaric precursor methods. To achieve accurate quantification, it needs high resolution mass data with low noise levels together with a powerful software able to correlate MS/MS identifications with full scan MS data and extract integrated ion intensities for the precursors and cognate, but often unidentified labeled analogues. We propose an alternative SILAC scheme that combines some useful features of iTRAQ-like work flows with the convenience of SILAC for studying samples from cultured cells. The technique is based on the incorporation of isobaric labeled amino acids that generate distinct residue immonium ion fragments. We show that reliable quantification is possible purely on MS/MS spectra using very simple data extraction tools. Unlabeled cell extracts were obtained from BJAB human B cells grown in standard RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin-streptomycin (all from Invitrogen). Metabolic labeling was performed on two human melanoma cell lines: SBCL2, derived from an radial growth phase melanoma (19Hsu M.Y. Shih D.T. Meier F.E. Van Belle P. Hsu J.Y. Elder D.E. Buck C.A. Herlyn M. Adenoviral gene transfer of β3 integrin subunit induces conversion from radial to vertical growth phase in primary human melanoma.Am. J. Pathol. 1998; 153: 1435-1442Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar) and kindly provided by Dr. Giovanella (Stehlin Foundation for Cancer Research, Houston, TX), and SKMel28, derived from skin metastasis (20Satyamoorthy K. DeJesus E. Linnenbach A.J. Kraj B. Kornreich D.L. Rendle S. Elder D.E. Herlyn M. Melanoma cell lines from different stages of progression and their biological and molecular analyses.Melanoma Res. 1997; 7: S35-S42PubMed Google Scholar) and obtained from the ATCC collection (number HTB-72, LGC Promochem). SBCL2 and SKmel28 cells were grown in high glucose Dulbecco's modified Eagle's medium deficient in l-valine, l-leucine, l-isoleucine, and l-glutamine (Cell Culture Technologies, Gravesano, Switzerland). The medium was reconstituted from concentrated stocks according to the manufacturer's instructions, sterile filtered, and stored at 4 °C. l-Isoleucine, l-leucine, and l-valine labeled either with 1-13C (99% 1-13C enrichment) or 15N (98% 15N enrichment) were obtained from Cambridge Isotope Laboratories (Andover, MA). The powdered amino acids were diluted in PBS to obtain stock solutions of 40 mg/ml for valine and 15 mg/ml for leucine and isoleucine that were sterile filtered and stored at −80 °C. SKMel28 and SBCL2 cells were grown in the base medium supplemented with 4 mm l-glutamine (Amimed, Allschwil, Switzerland), 10% dialyzed fetal bovine serum (Invitrogen), penicillin-streptomycin (Invitrogen), and the amino acids 1-13C- (for SKMel28) or 15N-labeled (for SBCL2 and SKMel28 as specified under "Results") l-valine, l-leucine, and l-isoleucine. The final concentrations of Val, Leu, and Ile were 94, 105, and 105 mg/liter, respectively, corresponding to the standard composition of the Dulbecco's modified Eagle's medium. Cell lines were grown for 3 weeks to achieve complete labeling. Cells at about 80% confluence were washed twice with PBS to remove serum proteins. Cells were detached by trypsinization and washed twice in PBS. Pellets of cells were resuspended in ice-cold hypotonic buffer (10 mm HEPES, 1.5 mm MgCl2, and 10 mm KCl) and sonicated for three cycles of 5 s each on ice. The lysates were centrifuged twice (10 min at 13,000 rpm at 4 °C) to pellet cellular debris; each time the supernatant was recovered. The protein concentration of the second supernatant was determined using the Bradford protein assay (Bio-Rad); the relative protein concentration of samples to be mixed was verified by densitometry on whole lanes of SDS-PAGE gels after Coomassie staining. For mixing experiments, lysates were combined in a known ratio equal to 1:3 for 15N:13C SKMel28 extracts or 1:1 for 13C SKMel28:15N SBCL2. For the co-elution experiment, 15N:13C SKMel28 lysates were combined in 1:2 ratio. 2D DIGE experiments were performed according to the manufacturer's instructions (GE Healthcare) (21Unlu M. Morgan M.E. Minden J.S. Difference gel electrophoresis: a single gel method for detecting changes in protein extracts.Electrophoresis. 1997; 18: 2071-2077Crossref PubMed Scopus (1859) Google Scholar). Briefly 300 μg of protein from 15N SBCL2 and 13C SKMel28 cell lysates were precipitated with acetone overnight at −20 °C; the pellet was dissolved in 25 μl of DIGE Cell Lysis Buffer (30 mm Tris/HCl, pH 8.5, 7 m urea, 2 m thiourea, 4% (w/v) CHAPS). 50 μg of protein from 15N SBCL2 and 13C SKMel28 lysates were minimally labeled with 800 pmol of reconstituted Cy3 and Cy5 dyes, respectively. 25 μg of the two samples were mixed together, labeled with Cy2, and used as internal standard. The Cy2-, Cy3-, and Cy5-labeled samples were combined, supplemented with 2% pH 3–11 ampholytes (IPG Buffer, GE Healthcare), and applied by cup-loading on a 13-cm, pH 3–11 non-linear Immobiline DryStrip (GE Healthcare) previously rehydrated in DIGE Cell Lysis Buffer overnight. Isoelectric focusing was performed on an Ettan IPGphor (GE Healthcare) system to attain a total of 22,500 V-h. Prior to the second dimension, strips were equilibrated for 15 min in a reducing buffer containing 6 m urea, 2% (w/v) SDS, 30% (v/v) glycerol, 32 mm DTT, 100 mm Tris, pH 8. This was followed by a 15-min alkylation in a buffer containing 6 m urea, 2% (w/v) SDS, 30% (v/v) glycerol, 240 mm iodoacetamide, 100 mm Tris, pH 8. Second dimension migration was carried out on a 13 × 9-cm 8–16% Criterion precast gel (Bio-Rad) at a constant voltage of 80 V for 4 h. The gel was rinsed in water and then scanned using a Molecular Imager FX (Bio-Rad) scanner. Gel images were analyzed using ImageMaster 2D Platinum DIGE Software version 5.0 (GE Healthcare). The gel was Coomassie-stained overnight and destained with 10% acetic acid. Spots of interest were manually excised from the gel. For 1D gel electrophoresis, 60 μg of protein from either pure or mixed lysates were subjected to limited electrophoretic separation on a 10% SDS-PAGE minigel, i.e. the migration was stopped after the front had moved by about 2.5 cm into the separating gel at which point the bands of a prestained size marker were visible and separated in the 20–250-kDa range. After Coomassie staining, each lane was cut into four fractions corresponding to regions of different molecular weights. Slices excised from 1D or 2D gels were transferred to 96-well plates. In-gel proteolytic cleavage with sequencing grade trypsin (Promega, Madison, WI) was performed automatically in a ProGest robotic work station (Genomic Solutions, Ann Arbor, MI) according to a described protocol (22Wilm M. Shevchenko A. Houthaeve T. Breit S. Schweigerer L. Fotsis T. Mann M. Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry.Nature. 1996; 379: 466-469Crossref PubMed Scopus (1507) Google Scholar) The liquid supernatant from the digestion was recovered and concentrated by evaporation. The final volume was adjusted to 60 μl with 0.1% TFA in 2% acetonitrile. Digests were desalted using C18 StageTips (Proxeon, Odense, Denmark), which have an estimated loading capacity equal to 10 μg. The peptides were eluted from the microcolumns with 80% acetonitrile after which the eluate was dried and resuspended in 7 μl of solvent A (2% acetonitrile, 0.5% formic acid). 2.5 μl of purified sample were injected on a reversed-phase C18 column (PepMap100, 3 μm, 100 Å, LC Packings) and separated by nanoflow liquid chromatography on an Ultimate (LC Packings) system on line with an electrospray quadrupole-time-of-flight mass spectrometer (API QSTAR Pulsar i, Applied Biosystems/SCIEX, Concord, Ontario, Canada). The gradient used for separation was from 2 to 40% acetonitrile in water with 0.5% formic acid at a flow rate of 200 nl/min. The mass spectrometer was controlled by the Analyst QS 1.1 software set to operate in information-dependent acquisition mode to automatically switch between MS and collision-induced dissociation MS/MS. Survey full-scan MS spectra were acquired from 400 to 1200 m/z in 1 s after which the two most intense ions with charge 2+ to 4+ were isolated for fragmentation. In a second method (method B), the three most intense ions with 2+ to 4+ charge were fragmented. Each parent ion underwent two different MS/MS acquisitions. The first scan (1-s duration) spanned the 50–1200 m/z mass range, used a collision energy proportional to precursor mass, and was intended to collect sequence information. The second MS/MS scan (0.2-s duration) was focused on (iso)leucine and valine immonium ions and therefore covered narrow ranges of masses (71–74 and 85–88 m/z for method "A" or a single scan at 64–95 m/z for method "B") to achieve a better sampling of the reporter ions. The collision energy for this narrow range scan was fixed at 60 eV to promote a higher degree of fragmentation, and the masses were acquired in the "enhanced" mode that allows a considerable gain in signal intensity by specifically pulsing ions of a narrow mass range (23Chernushevich I.V. Loboda A.V. Thomson B.A. An introduction to quadrupole-time-of-flight mass spectrometry.J. Mass Spectrom. 2001; 36: 849-865Crossref PubMed Scopus (548) Google Scholar). Three (method A) or two (method B) MS/MS spectra were cumulated for each selected peptide; former target peptides and their isotopes were dynamically excluded for 90 s (with a mass tolerance of 50 milli-mass units). All runs were performed in triplicate for the 15N SKMel28:13C SKMel28 1:3 mixture fraction using method A. The 15N SBCL2:13C SKMel28 1:1 mixture was analyzed in quadruplicate using both methods A and B in duplicate. For the analysis of 2D DIGE spots by LC-MS a shorter gradient from 2 to 40% acetonitrile in 45 min was used. All MS parameters were the same as those described above. For testing peptide co-elution, a special acquisition method was used in which two precursors were fragmented for 10 consecutive scans, giving a total cycle time of 21 s. The elution profile of the peak was then followed to monitor immonium ion fragment intensities during the same cycle. The Mascot.dll script (version 1.6b21) supplied by Matrix Science was used to extract spectra of each run using the following parameters: 2+ to 4+ charge state, peak centroiding, no deisotoping, spectra rejected if containing less than eight peaks, no minimum peak intensity required, report peak area. For mixing experiments, Mascot generic format flat text (.mgf) files deriving from different fractions of the same mixture were pooled before performing the database search. Proteins were identified using Mascot version 2.1 (Matrix Science, London, UK) and searching the UniProt database, restricted to human taxonomy. Database releases used were 8.7 of September, 19 2006 for experiments with unlabeled samples (71,233 sequences after taxonomy filter) and release 11.0 of May, 29 2007 (70,004 sequences after taxonomy filter) for experiments with labeled samples. One trypsin missed cleavage was allowed, cysteine carbamidomethylation was set as fixed modification, and methionine oxidation was defined as variable modification. Unless otherwise specified, peptide mass tolerance was set to 1.2 Da (0.6 Da for unlabeled samples), and the fragment mass tolerance was set to 0.3 Da. We used a broad peptide mass tolerance (1.2 Da) to include in Mascot version 2.1 searches the +1-Da 13C precursor peptides that can be incorrectly chosen during peak detection. For labeled samples, 13C labeling and 15N labeling on valine, isoleucine, and leucine were also set as fixed peptide modifications unless specified. The Mascot multidimensional protein identification technology (MudPIT) scoring system was used for all analyses of mixtures (the unlabeled sample was analyzed with standard scoring) with significance threshold at p = 0.05 and minimum ion score equal to 14. The same .mgf files used for protein identification mentioned above were searched against a decoy database automatically generated by Mascot version 2.2 and containing random sequences having the same length and average amino acid composition of the forward "normal" database to compute the false discovery rate (FDR) of protein identification. Significance threshold was set at p = 0.05, and minimum ion score was set at 14 as in the previous search used for protein identification; the resulting FDR values are reported under "Results" for the different shotgun analyses. Proteins spanning the same set of peptides, or a subset, were collapsed into a single entry on the hit list according to the principle of parsimony. For these cases, only one representative protein is listed. Proteins with matched peptides in common with other sequences were validated only if matched by at least one unique ("bold red") discriminating peptide. No further attempt was made to discriminate protein isoforms that were indistinguishable with the available mass spectrometry data. To obtain protein quantification, an in-house built Perl (version 5.2.2.0) script extracted the intensity values of the following masses from the Mascot .dat result file: 72.081 amu for [13C]valine, 73.078 amu for [15N]valine, 86.097 amu for [13C](iso)leucine, and 86.094 amu for [15N](iso)leucine with a tolerance of ±0.020 amu. For each identified protein, these intensities were associated to the corresponding valine- and (iso)leucine-containing peptides. In the case of peptides containing both valine and (iso)leucine, the quantification was considered as independent. Only Val-containing peptides with non-zero values on both 72.081 and 73.078 m/z and Ile/Leu-containing peptides with non-zero values on both 86.097 and 87.094 m/z were used for estimation of abundance ratios. R software (24R Development Core TeamR: a Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria2005Google Scholar) (2.3.1 version) was used for all statistical calculations. For each protein presenting at least four quantified peptides, a straight line was fitted by least squares through the points represented by the 13C and 15N immonium ion intensities for each peptide; the corresponding slope is an estimate of the abundance ratio (25Pan C. Kora G. Tabb D.L. Pelletier D.A. McDonald W.H. Hurst G.B. Hettich R.L. Samatova N.F. Robust estimation of peptide abundance ratios and rigorous scoring of their variability and bias in quantitative shotgun proteomics.Anal. Chem. 2006; 78: 7110-7120Crossref PubMed Scopus (38) Google Scholar). The standard error of the estimate, the t value of the test for significance of the slope, and the corresponding p value as well as the 95% confidence interval were also calculated from the linear regression. Outlier points, which could result either from experimental variability or from the presence of differentially expressed isoforms of the same protein matched by the same set of peptides, were not removed before linear regression. Thus no attempt was made to discriminate such protein isoforms. The regression line was fitted twice using in turn the 13C and 15N immonium ion intensities as response variable; the two estimations of the abundance ratio were extremely close, and the one that produced the smallest residuals was used. Other methods were considered in particular orthogonal regression, which produced extremely similar results (data not shown). Because the ratio 15N/13C should in theory remain constant for all peptides regardless of the intensities of the individual ions, the regression line was constrained to pass through the origin. Examination of the plots for proteins with a large number of peptides (the first 30 Mascot protein matches in the 1:1 13C SKMel28:15N SBCL2 data set) suggested that this assumption is correct. Unless otherwise specified, the reported ratios are defined heavy/light (H/L). Isobaric SILAC with immonium ion splitting (ISIS) differential labeling is based on the incorporation of isobaric analogues of specific amino acids capable of providing distinct immonium ions fragments whose intensity can be relatively quantified in MS/MS spectra. Essential amino acids containing the stable 1-13C (culture A) or α-15N (culture B) isotope are added to a cell culture medium lacking these same amino acids but containing all others in unlabeled form (Fig. 1A). Cells are then grown for a time sufficient to obtain a near complete (98% or more) protein labeling, i.e. six or more cell divisions. Every labeled amino acid residue (be it 1-13C- or α-15N-labeled) in a peptide or protein increases its nominal mass by 1 amu. Corresponding peptides originating from the cultures A and B should therefore be isobaric, but they are expected to generate upon collision-induced fragmentation different immonium ions. These residue ions are produced by the simultaneous cleavage of both the peptide bond N-terminal to the residue of interest as well as of the Cα–CO bond within the residue (26Falick A.M. Hines W.M. Medzihradszky K.F. Baldwin M.A. Gibson B.W. Low-mass ions produced from peptides by high-energy collision-induced dissociation in tandem mass spectrometry.J. Am. Soc. Mass Spectrom. 1993; 4: 882-893Crossref PubMed Scopus (160) Google Scholar). Residues labeled with 13C on the carbonyl carbon (position 1) thus produce "light" immonium ions identical to those generated by naturally occurring (unlabeled) residues because the labeled C-1 atom is lost during MS/MS fragmentation. On the other hand, amino acid residues labeled on the α carbon or 15N-labeled on the backbone nitrogen are expected to produce immonium ions 1 amu heavier than the natural ones because the label is retained after CID. To perform relative protein quantification, extracts from differentially labeled cells can be mixed directly after lysis, preserving in this way the ratio of 1-13C- to α-15N-labeled proteins during the next steps of sample preparation (Fig. 1B). The protein mixture is then digested, and the resulting peptides are analyzed by LC-MS/MS. The intensity values of the couples of immonium ions deriving from differentially la