Methods for Peptide and Protein Quantitation by Liquid Chromatography-Multiple Reaction Monitoring Mass Spectrometry
2011; Elsevier BV; Volume: 10; Issue: 6 Linguagem: Inglês
10.1074/mcp.m110.006593
ISSN1535-9484
AutoresHaixia Zhang, Qinfeng Liu, Lisa J. Zimmerman, Amy‐Joan L. Ham, Robbert J.C. Slebos, Jamshedur Rahman, Takefume Kikuchi, Pierre P. Massion, David P. Carbone, Dean Billheimer, D.C. Liebler,
Tópico(s)Enzyme Structure and Function
ResumoLiquid chromatography-multiple reaction monitoring mass spectrometry of peptides using stable isotope dilution (SID) provides a powerful tool for targeted protein quantitation. However, the high cost of labeled peptide standards for SID poses an obstacle to multiple reaction monitoring studies. We compared SID to a labeled reference peptide (LRP) method, which uses a single labeled peptide as a reference standard for all measured peptides, and a label-free (LF) approach, in which quantitation is based on analysis of un-normalized peak areas for detected MRM transitions. We analyzed peptides from the Escherichia coli proteins alkaline phosphatase and β-galactosidase spiked into lysates from human colon adenocarcinoma RKO cells. We also analyzed liquid chromatography-multiple reaction monitoring mass spectrometry data from a recently published interlaboratory study by the National Cancer Institute Clinical Proteomic Technology Assessment for Cancer network (Addona et al. (2009) Nat. Biotechnol. 27: 633–641), in which unlabeled and isotopically labeled synthetic peptides or their corresponding proteins were spiked into human plasma. SID displayed the highest correlation coefficients and lowest coefficient of variation in regression analyses of both peptide and protein spike studies. In protein spike experiments, median coefficient of variation values were about 10% for SID and 20–30% for LRP and LF methods. Power calculations indicated that differences in measurement error between the methods have much less impact on measured protein expression differences than biological variation. All three methods detected significant (p < 0.05) differential expression of three endogenous proteins in a test set of 10 pairs of human lung tumor and control tissues. Further, the LRP and LF methods both detected significant differences (p < 0.05) in levels of seven biomarker candidates between tumors and controls in the same set of lung tissue samples. The data indicate that the LRP and LF methods provide cost-effective alternatives to SID for many quantitative liquid chromatography-multiple reaction monitoring mass spectrometry applications. Liquid chromatography-multiple reaction monitoring mass spectrometry of peptides using stable isotope dilution (SID) provides a powerful tool for targeted protein quantitation. However, the high cost of labeled peptide standards for SID poses an obstacle to multiple reaction monitoring studies. We compared SID to a labeled reference peptide (LRP) method, which uses a single labeled peptide as a reference standard for all measured peptides, and a label-free (LF) approach, in which quantitation is based on analysis of un-normalized peak areas for detected MRM transitions. We analyzed peptides from the Escherichia coli proteins alkaline phosphatase and β-galactosidase spiked into lysates from human colon adenocarcinoma RKO cells. We also analyzed liquid chromatography-multiple reaction monitoring mass spectrometry data from a recently published interlaboratory study by the National Cancer Institute Clinical Proteomic Technology Assessment for Cancer network (Addona et al. (2009) Nat. Biotechnol. 27: 633–641), in which unlabeled and isotopically labeled synthetic peptides or their corresponding proteins were spiked into human plasma. SID displayed the highest correlation coefficients and lowest coefficient of variation in regression analyses of both peptide and protein spike studies. In protein spike experiments, median coefficient of variation values were about 10% for SID and 20–30% for LRP and LF methods. Power calculations indicated that differences in measurement error between the methods have much less impact on measured protein expression differences than biological variation. All three methods detected significant (p < 0.05) differential expression of three endogenous proteins in a test set of 10 pairs of human lung tumor and control tissues. Further, the LRP and LF methods both detected significant differences (p < 0.05) in levels of seven biomarker candidates between tumors and controls in the same set of lung tissue samples. The data indicate that the LRP and LF methods provide cost-effective alternatives to SID for many quantitative liquid chromatography-multiple reaction monitoring mass spectrometry applications. A rapidly evolving approach to protein quantitation is the targeted analysis of representative peptides by liquid chromatography-tandem mass spectrometry by multiple reaction monitoring (LC-MRM-MS) 1The abbreviations used are:LC-MRM-MSliquid chromatography-tandem mass spectrometry by multiple reaction monitoringSIDstable isotope dilutionLRPlabeled reference peptideLFlabel-freeCPTACClinical Proteomic Technology Assessment for CancerADCadeno carcinomaSCCsquamous cell carcinomaQCquality controlAICAkaike's information criterionCVcoefficient of varianceRSrelative sensitivity. analysis (1Rifai N. Gillette M.A. Carr S.A. Protein biomarker discovery and validation: the long and uncertain path to clinical utility.Nat. Biotechnol. 2006; 24: 971-983Crossref PubMed Scopus (1371) Google Scholar, 2Anderson L. Hunter C.L. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins.Mol. Cell Proteomics. 2006; 5: 573-588Abstract Full Text Full Text PDF PubMed Scopus (1081) Google Scholar, 3Addona T.A. Abbatiello S.E. Schilling B. Skates S.J. Mani D.R. Bunk D.M. Spiegelman C.H. Zimmerman L.J. Ham A.J. Keshishian H. Hall S.C. Allen S. Blackman R.K. Borchers C.H. Buck C. Cardasis H.L. Cusack M.P. Dodder N.G. Gibson B.W. Held J.M. Hiltke T. Jackson A. Johansen E.B. Kinsinger C.R. Li J. Mesri M. Neubert T.A. Niles R.K. Pulsipher T.C. Ransohoff D. Rodriguez H. Rudnick P.A. Smith D. Tabb D.L. Tegeler T.J. Variyath A.M. Vega-Montoto L.J. Wahlander A. Waldemarson S. Wang M. Whiteaker J.R. Zhao L. Anderson N.L. Fisher S.J. Liebler D.C. Paulovich A.G. Regnier F.E. Tempst P. Carr S.A. Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma.Nat. Biotechnol. 2009; 27: 633-641Crossref PubMed Scopus (867) Google Scholar). In this approach, peptides are quantified by monitoring several MRM transitions for each peptide with either a triple quadrupole or a quadrupole-ion trap instrument. Stable isotope dilution (SID), in which labeled peptides are used as internal standards is considered the gold standard for rigorous quantitation by LC-MRM-MS (1Rifai N. Gillette M.A. Carr S.A. Protein biomarker discovery and validation: the long and uncertain path to clinical utility.Nat. Biotechnol. 2006; 24: 971-983Crossref PubMed Scopus (1371) Google Scholar, 4Gerber S.A. Rush J. Stemman O. Kirschner M.W. Gygi S.P. Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 6940-6945Crossref PubMed Scopus (1549) Google Scholar, 5Kirkpatrick D.S. Gerber S.A. Gygi S.P. The absolute quantification strategy: a general procedure for the quantification of proteins and post-translational modifications.Methods. 2005; 35: 265-273Crossref PubMed Scopus (484) Google Scholar). In contrast to antibody-based quantitation, where antibody availability and specificity are often limiting, LC-MRM-MS enables configuration of an assay for essentially any protein. In practice, this approach has proven sensitive enough to apply to challenging protein quantitation problems. For example, proteins can be quantified at single-digit copy numbers in cells (6Picotti P. Bodenmiller B. Mueller L.N. Domon B. Aebersold R. Full dynamic range proteome analysis of S. cerevisiae by targeted proteomics.Cell. 2009; 138: 795-806Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar) and in plasma at levels approaching ng/ml (7Kuzyk M.A. Smith D. Yang J. Cross T.J. Jackson A.M. Hardie D.B. Anderson N.L. Borchers C.H. Multiple reaction monitoring-based, multiplexed, absolute quantitation of 45 proteins in human plasma.Mol. Cell Proteomics. 2009; 8: 1860-1877Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar, 8Keshishian H. Addona T. Burgess M. Mani D.R. Shi X. Kuhn E. Sabatine M.S. Gerszten R.E. Carr S.A. Quantification of cardiovascular biomarkers in patient plasma by targeted mass spectrometry and stable isotope dilution.Mol. Cell Proteomics. 2009; 8: 2339-2349Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). With antibody-based enrichment, LC-MRM-MS can achieve even greater sensitivity (9Whiteaker J.R. Zhao L. Anderson L. Paulovich A.G. An automated and multiplexed method for high throughput peptide immunoaffinity enrichment and multiple reaction monitoring mass spectrometry-based quantification of protein biomarkers.Mol. Cell Proteomics. 2009; 10 (M110.005645, E-pub)Google Scholar, 10Anderson N.L. Anderson N.G. Haines L.R. Hardie D.B. Olafson R.W. Pearson T.W. Mass spectrometric quantitation of peptides and proteins using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA).J. Proteome Res. 2004; 3: 235-244Crossref PubMed Scopus (696) Google Scholar, 11Anderson N.L. Jackson A. Smith D. Hardie D. Borchers C. Pearson T.W. SISCAPA peptide enrichment on magnetic beads using an in-line bead trap device.Mol. Cell Proteomics. 2009; 8: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 12Hoofnagle A.N. Becker J.O. Wener M.H. Heinecke J.W. Quantification of thyroglobulin, a low-abundance serum protein, by immunoaffinity peptide enrichment and tandem mass spectrometry.Clin. Chem. 2008; 54: 1796-1804Crossref PubMed Scopus (242) Google Scholar). liquid chromatography-tandem mass spectrometry by multiple reaction monitoring stable isotope dilution labeled reference peptide label-free Clinical Proteomic Technology Assessment for Cancer adeno carcinoma squamous cell carcinoma quality control Akaike's information criterion coefficient of variance relative sensitivity. Despite the power of the method, the use of SID is nevertheless limited practically by the cost of labeled standards, which are expensive (∼$1000 per milligram for labeled peptides of high purity). This issue is particularly important in considering LC-MRM-MS to evaluate candidate biomarkers for disease. Application of biomarker discovery platforms, such as shotgun proteomics or transcriptome profiling can yield hundreds of biomarker candidates. The next phase of analysis, termed "verification," consists of configuring assays for the candidates and evaluating them in well-defined test cohorts (1Rifai N. Gillette M.A. Carr S.A. Protein biomarker discovery and validation: the long and uncertain path to clinical utility.Nat. Biotechnol. 2006; 24: 971-983Crossref PubMed Scopus (1371) Google Scholar). The cost of configuring SID-LC-MRM-MS assays for three representative peptides each for 50 proteins would be approximately $150,000. An MRM-based approach for targeted protein quantitation with a more limited number of isotopically labeled standards could be particularly useful for biomarker candidate screening, in which the expense of labeled standards presents a real barrier to verification of large numbers of candidates. Although SID should outperform methods that do not employ labeled standards for each analyte, there are insufficient data available to evaluate the performance of alternative techniques or to determine appropriate contexts for their use. Here we compared SID with two alternative methods. The first is a labeled reference peptide (LRP) method, which employs a single isotopically labeled peptide as the reference peptide for all of the other peptide analytes. The second is a label-free (LF) method that employs no standard and where quantitation is based only on the peak areas extracted from LC-MRM-MS product ion chromatograms. We compared these three approaches with datasets from analyses of defined peptide and protein mixtures on triple quadrupole LC-MS instruments. Test samples included synthetic peptides or their corresponding proteins spiked into a human cell lysate. We also analyzed LC-MRM-MS data from a recent study by the National Cancer Institute Clinical Proteomic Technology Assessment for Cancer (CPTAC) program (3Addona T.A. Abbatiello S.E. Schilling B. Skates S.J. Mani D.R. Bunk D.M. Spiegelman C.H. Zimmerman L.J. Ham A.J. Keshishian H. Hall S.C. Allen S. Blackman R.K. Borchers C.H. Buck C. Cardasis H.L. Cusack M.P. Dodder N.G. Gibson B.W. Held J.M. Hiltke T. Jackson A. Johansen E.B. Kinsinger C.R. Li J. Mesri M. Neubert T.A. Niles R.K. Pulsipher T.C. Ransohoff D. Rodriguez H. Rudnick P.A. Smith D. Tabb D.L. Tegeler T.J. Variyath A.M. Vega-Montoto L.J. Wahlander A. Waldemarson S. Wang M. Whiteaker J.R. Zhao L. Anderson N.L. Fisher S.J. Liebler D.C. Paulovich A.G. Regnier F.E. Tempst P. Carr S.A. Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma.Nat. Biotechnol. 2009; 27: 633-641Crossref PubMed Scopus (867) Google Scholar), which analyzed human plasma spiked with peptide and protein standards. Finally, we compared the methods in analysis of several lung cancer biomarker candidate proteins in normal lung and lung-tumor tissues. Our studies document the performance of all three methods with the same datasets. The data establish the performance of the LRP and LF methods and provide a basis to select between all three methods for appropriate applications in quantitative proteomics. Iodoacetamide and ammonium bicarbonate (>99.0% purity) were from Sigma (St. Louis, MO); d,l-1,4 dithiothreitol was from Bio-Rad (Hercules, CA); 2,2,2-trifluoroethanol was from Acros (Geel, Belgium). Two Escherichia coli proteins, alkaline phosphatase (AP) and β-galactosidase (BG) were purchased from Sigma. Six pairs of C-terminal isotopically labeled peptides containing U-13C6, U-15N4-arginine or U-13C6, U-15N2-lysine and corresponding unlabeled peptides derived from AP and BG were supplied by New England Peptide, LLC (Gardner, MA) at over 95% chemical purity according to amino acid analysis (shown in Table I). Three C-terminal isotopically labeled peptides containing U-13C6, U-15N4-arginine or U-13C6, U-15N2-lysine from human advanced glycosylation end product-specific receptor (AGER‖‖) (VLSPQGGGPWDSVA*R), from γ- and β-actin (referred to herein as ACTIN to denote both proteins) (GYSFTTTAE*R) and from annexin A1 (ANXA1) (VLDLEL*K) also were obtained from New England Peptide at 95% chemical purity. Mass spectrometry grade trypsin (Trypsin Gold) was purchased from Promega (Madison, WI). HPLC grade water and acetonitrile were from Mallinckrodt Baker (Phillipsburg, NJ).Table IAP and BG peptides and transitions selected for LC-MRM MSProteinPeptideaPeptides marked with * are isotopically labeled.Precursor m/zProduct m/zβ-galactosidase (BG)LPSEFDLSAFLR (BG 698)697.9593.34, 706.42, 821.45, 968.52LPSEFDLSAFL*R702.9603.35, 716.43, 831.46, 978.53LWSAEIPNLYR (BG 681)681.4662.36, 775.45, 904.49, 1062.56LWSAEIPNLY*R686.4672.37, 785.45, 914.50, 1072.57APLDNDIGVSEATR (BG 729)729.4563.28, 719.37, 832.45, 1061.52APLDNDIGVSEAT*R734.4573.29, 729.38, 842.46, 1071.53Alkaline phosphatase (AP)AAQGDITAPGGAR (AP 593)592.8457.25, 528.29, 629.34, 914.47AAQGDITAPGGA*R597.8467.26, 538.30, 639.34, 924.48APGLTQALNTK (AP 557)557.3546.32, 674.38, 775.43, 945.54APGLTQALNT*K561.3554.34, 682.40, 783.44, 953.55NYAEGAGGFFK (AP 581)580.8555.29, 683.35, 812.39, 883.43NYAEGAGGFF*K584.8563.31, 691.36, 820.41, 891.44a Peptides marked with * are isotopically labeled. Open table in a new tab The human colon adenocarcinoma cell line (RKO) was cultured at 37 °C in the McCoy's 5A medium (Mediatech, Herndon, VA) with 10% fetal bovine serum (Atlas Biologicals, Fort Collins, CO) in the presence of 5% CO2 and harvested at >85% confluence. Cells were washed twice with 10 ml phosphate-buffered saline, collected in 10 ml phosphate-buffered saline buffer, and then centrifuged at 2000 rpm for 5 min at 4 °C to obtain the cell pellet. Cells were lysed and proteins were extracted with ammonium bicarbonate and 2,2,2-trifluoroethanol as described previously (13Slebos R.J. Brock J.W. Winters N.F. Stuart S.R. Martinez M.A. Li M. Chambers M.C. Zimmerman L.J. Ham A.J. Tabb D.L. Liebler D.C. Evaluation of strong cation exchange versus isoelectric focusing of peptides for multidimensional liquid chromatography-tandem mass spectrometry.J. Proteome Res. 2008; 7: 5286-5294Crossref PubMed Scopus (80) Google Scholar). Protein concentration of cell lysate was measured using bicinchoninic acid assay with bovine serum albumin used as protein standard. Three peptides each from AP and BG were synthesized for spike studies and are listed in Table I together with their corresponding isotopically labeled standards. The AP and BG peptides were spiked into an RKO cell lysate, which then was processed by a workflow based on in-solution tryptic digestion. The protein concentration of RKO lysate or the digest was 0.5 μg/μl. The unlabeled AP and BG peptides were spiked in at equal molarity at 2, 10, 40, 100, or 200 fmol/μg protein. Each isotopically labeled standard peptide was spiked at a constant concentration of 60 fmol/μg protein. Two samples were prepared and processed for each experimental variation and concentration for LC-MRM-MS (see below). The RKO cell lysate was subjected to tryptic digestion by a modification of a previously reported method (13Slebos R.J. Brock J.W. Winters N.F. Stuart S.R. Martinez M.A. Li M. Chambers M.C. Zimmerman L.J. Ham A.J. Tabb D.L. Liebler D.C. Evaluation of strong cation exchange versus isoelectric focusing of peptides for multidimensional liquid chromatography-tandem mass spectrometry.J. Proteome Res. 2008; 7: 5286-5294Crossref PubMed Scopus (80) Google Scholar). Briefly, proteins were reduced with 10 mm dithiotreitol for 30 min at 65 °C and then alkylated with 20 mm iodoacetamide for 30 min in the dark at room temperature, followed by the addition of trypsin with 1:50 enzyme to protein ratio. Digestion was performed at 37 °C overnight and was terminated with the addition of formic acid (>98% purity, EMD, Darmstadt, Germany) to a final concentration of 1%. The peptide mixture was desalted with an Oasis HLB extraction plate (Waters Corp., Milford, MA), which was prewashed with 1 ml of acetonitrile and then equilibrated with 2 ml of water. Following sample loading, plates were washed with 1 ml water and the peptides were eluted with 80% aqueous acetonitrile. The eluate then was evaporated in a SpeedVac concentrator (Thermo-Fisher, Waltham, MA) and the peptides were reconstituted in water containing 0.1% formic acid for LC-MRM-MS analysis. A second set of experiments employed AP and BG proteins spiked an RKO lysate (0.5 μg/μl protein) background. AP and BG were added at equimolar concentrations of 5, 20, 80, and 200 fmol/μg protein. Three synthetic isotopically labeled peptides each from AP and BG were spiked into each protein mixture to achieve a concentration of 60 fmol/μg protein and in-solution tryptic digestion and peptide recovery and desalting were performed as described above. Five replicates were prepared for each spike concentration and 2 μl was injected on-column for LC-MRM-MS. LC-MRM-MS analyses were done as part of a CPTAC interlaboratory study (3Addona T.A. Abbatiello S.E. Schilling B. Skates S.J. Mani D.R. Bunk D.M. Spiegelman C.H. Zimmerman L.J. Ham A.J. Keshishian H. Hall S.C. Allen S. Blackman R.K. Borchers C.H. Buck C. Cardasis H.L. Cusack M.P. Dodder N.G. Gibson B.W. Held J.M. Hiltke T. Jackson A. Johansen E.B. Kinsinger C.R. Li J. Mesri M. Neubert T.A. Niles R.K. Pulsipher T.C. Ransohoff D. Rodriguez H. Rudnick P.A. Smith D. Tabb D.L. Tegeler T.J. Variyath A.M. Vega-Montoto L.J. Wahlander A. Waldemarson S. Wang M. Whiteaker J.R. Zhao L. Anderson N.L. Fisher S.J. Liebler D.C. Paulovich A.G. Regnier F.E. Tempst P. Carr S.A. Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma.Nat. Biotechnol. 2009; 27: 633-641Crossref PubMed Scopus (867) Google Scholar), in which both peptide-spike and protein-spike experiments were done. In the peptide-spike experiment (Study I), 11 synthetic peptides derived from seven proteins (bovine aprotinin, murine leptin, equine myoglobin, bovine myelin basic protein, human prostate specific antigen horseradish peroxidase, and human C-reactive protein) were spiked into digested human plasma (1 μg/μl) at concentrations ranging from 1 to 500 fmol/μl. An equimolar mixture of 11 stable-isotope labeled peptides corresponding to the seven proteins (three of the proteins were represented by multiple peptides) was spiked in at 50 fmol/μl as internal standards. In the protein-spike experiment (Study III), the seven intact proteins were spiked into undigested plasma and the mixture was then subjected to reduction, alkylation, and digestion, followed by addition of the internal standards. For LC-MRM-MS analyses, 1 μl was injected on-column. Three MRM transitions were recorded for each peptide and four replicates were collected for each concentration. Other details for sample preparation and experiment design of this work can be found in (3Addona T.A. Abbatiello S.E. Schilling B. Skates S.J. Mani D.R. Bunk D.M. Spiegelman C.H. Zimmerman L.J. Ham A.J. Keshishian H. Hall S.C. Allen S. Blackman R.K. Borchers C.H. Buck C. Cardasis H.L. Cusack M.P. Dodder N.G. Gibson B.W. Held J.M. Hiltke T. Jackson A. Johansen E.B. Kinsinger C.R. Li J. Mesri M. Neubert T.A. Niles R.K. Pulsipher T.C. Ransohoff D. Rodriguez H. Rudnick P.A. Smith D. Tabb D.L. Tegeler T.J. Variyath A.M. Vega-Montoto L.J. Wahlander A. Waldemarson S. Wang M. Whiteaker J.R. Zhao L. Anderson N.L. Fisher S.J. Liebler D.C. Paulovich A.G. Regnier F.E. Tempst P. Carr S.A. Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma.Nat. Biotechnol. 2009; 27: 633-641Crossref PubMed Scopus (867) Google Scholar). Surgically resected human lung tumor samples (adenocarcinomas (ADC), squamous cell carcinomas (SCC), and normal lung tissues dissected at least 2 cm from each tumor) were collected at Vanderbilt University Medical Center, snap frozen, and kept in liquid nitrogen until use for individual analysis. Informed consent was obtained and the project was approved by the Vanderbilt University Medical Center Institutional Review Board. Tissues were cut into small pieces in a Petri dish on dry ice. Ice cold RIPA buffer was then added to tissue pieces at a ratio of 100 mg/ml (w/v). Tissues were homogenized with a Brinkmann Polytron Homogenizer (Brinkmann, Switzerland) in RIPA buffer on ice. Lysates were left on ice for 30 min followed by centrifugation at 20,000 × g for 15 min at 4 °C. Supernatants were stored at −80 °C. Protein concentrations of the supernatants were estimated using bichinchoninic acid assay (Thermo Scientific, Rockford, IL) with bovine serum albumin used as a standard. Protein digests from lung tumor and normal tissue lysates were subjected to short (∼1 cm) SDS-PAGE separation, cleanup and in-gel digestion, as described previously (14Lapierre L.A. Avant K.M. Caldwell C.M. Ham A.J. Hill S. Williams J.A. Smolka A.J. Goldenring J.R. Characterization of immunoisolated human gastric parietal cells tubulovesicles: identification of regulators of apical recycling.Am. J. Physiol. Gastrointest Liver Physiol. 2007; 292: G1249-1262Crossref PubMed Scopus (59) Google Scholar). Tissue protein (20 μg) was prepared in 4× LDS buffer and loaded onto NuPAGE 10% Bis-Tris SDS-PAGE gel (Invitrogen, Carlsbad, CA). Isotopically labeled peptides from AGER, ANXA1, and ACTIN were spiked into solution at a concentration of 60 fmol/μg protein prior to in-gel digestion. Peptides were extracted as described above and reconstituted in 40 μl 0.1% formic acid for LC-MRM-MS analysis. Because of limited amount of available tissue, a single process replicate of each sample was analyzed. The selection of signature peptides was based on criteria reported previously (5Kirkpatrick D.S. Gerber S.A. Gygi S.P. The absolute quantification strategy: a general procedure for the quantification of proteins and post-translational modifications.Methods. 2005; 35: 265-273Crossref PubMed Scopus (484) Google Scholar, 15Keshishian H. Addona T. Burgess M. Kuhn E. Carr S.A. Quantitative, multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution.Mol. Cell Proteomics. 2007; 6: 2212-2229Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar, 16Li M. Gray W. Zhang H. Chung C.H. Billheimer D. Yarbrough W.G. Liebler D.C. Shyr Y. Slebos R.J. Comparative shotgun proteomics using spectral count data and quasi-likelihood modeling.J. Proteome Res. 2010; 9: 4295-4305Crossref PubMed Scopus (86) Google Scholar), which consider unique (proteotypic) peptide sequences and features that enhance chemical stability. Priority was given to those peptides that were previously identified in the shotgun data set with high MS/MS spectral quality. Previous work demonstrated a high correlation between intense product ions in ion trap MS/MS spectra and the most intense MRM transitions on triple quadrupole instruments (17Prakash A. Tomazela D.M. Frewen B. Maclean B. Merrihew G. Peterman S. Maccoss M.J. Expediting the development of targeted SRM assays: using data from shotgun proteomics to automate method development.J. Proteome Res. 2009; 8: 2733-2739Crossref PubMed Scopus (121) Google Scholar, 18Sherwood C.A. Eastham A. Lee L.W. Risler J. Vitek O. Martin D.B. Correlation between y-type ions observed in ion trap and triple quadrupole mass spectrometers.J. Proteome Res. 2009; 8: 4243-4251Crossref PubMed Scopus (47) Google Scholar). Additional peptides were selected by in silico digestion and all peptides included in MRM analysis were required to have 7 to 25 amino acids in length, be fully tryptic (both N- and C termini are formed by cleavage at lysine or arginine) and contain no ragged ends or potential post translational modification motifs (e.g. NXT/S, for possible N-glycosylation). Peptides containing cysteine or methionine residues were not excluded and cysteines were present as carboxyamidomethylated derivatives following treatment with iodoacetamide during sample work-up. Peptide uniqueness was confirmed by searching against the International Protein Index human database (Version 3.56). Criteria to include specific signature peptides and their MRM transitions for use in our experiments were as follows: (1) when peptide standards were used, chromatographic retention time alignment was required for both precursor signal and MRM transitions; (2) when standard peptides were available, the relative intensities of MRM transition signals were consistent with those observed in full scan MS/MS of the corresponding standards; (3) when no standard peptides were available, relative intensities of MRM transition signals were consistent with those observed previously in linear ion trap MS/MS spectra and were consistent between different samples; (4) in analyses of lung tissue samples, at least three of the specified MRM transitions with measured signal-to-noise greater than three were observed in either normal or tumor samples. Signal-to-noise was estimated as S/N=(b-a)/a(Eq. 1) where b is the signal intensity for the measured peak and a is the mean signal intensity over the intervals equal to ∼10 times peak width measured both immediately before and immediately following elution. Analyses for AP/BG spike experiments were performed on a TSQ Quantum triple quadrupole mass spectrometer (Thermo-Fisher Scientific, Waltham MA) equipped with an Eksigent 1D Plus NanoLC pump (Eksigent Technologies, Dublin CA). The mobile phase consisted of solvent A, 0.1% aqueous formic acid and solvent B, acetonitrile with 0.1% formic acid. Peptides were separated on a capillary column (Polymicro Technologies, 100 μm × 11 cm) packed with Jupiter C18 resin (5 μm, 300 Å, Phenomenex) using an in-line solid-phase extraction column (100 μm × 6 cm) packed with the same C18 resin (using a frit generated with liquid silicate Kasil 1 similar to that previously described (19Licklider L.J. Thoreen C.C. Peng J. Gygi S.P. Automation of nanoscale microcapillary liquid chromatography-tandem mass spectrometry with a vented column.Anal. Chem. 2002; 74: 3076-3083Crossref PubMed Scopus (186) Google Scholar). Injections were 2 μl of a sample solution containing 0.5 mg/ml peptide mixture (based on protein concentration) and were followed by a 10 min wash period with 100% A, then by elution with a gradient of 2–25% solvent B in 25 min, 25–50% solvent B in 20 min, and followed by 50–90% solvent B in 10 min. LC-MRM-MS analyses of the AP and BG peptide and protein spike samples were done with an electrospray voltage of 1200 V, capillary temperature 210 °C and skimmer offset −5 V. Both Q1 and Q3 were set at unit resolution (FWHM 0.7 Da) and collision gas (He) pressure in Q2 was held at 1.5 mTorr. Scan width was 0.004 m/z and scan time was 20 ms for the AP and BG peptide and protein analyses and 10 ms for lung tissue samples. Collision energy for each peptide was calculated based on the equation CE==0.034*m/z+3.314(Eq. 2) in which the m/z is the mass to charge ratio of the precursor ion. Peak areas for each peptide were extracted and integrated using Skyline software v. 0.6.1.2168 (20MacLean B. Tomazela D.M. Shulman N. Chambers M. Finney G.L. Frewen B. Kern R. Tabb D.L. Liebler D.C. MacCoss M.J. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments.Bioinformatics. 2010; 26: 966-968Crossref PubMed Scopus (3006) Google Scholar). Routine assessment of instrument and chromatographic performance was done with a quality control (QC) standard consisting of
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