Quantitative Profiling of Post-translational Modifications by Immunoaffinity Enrichment and LC-MS/MS in Cancer Serum without Immunodepletion

A robust method was developed and optimized for enrichment and quantitative analysis of posttranslational modifications (PTMs) in serum/plasma samples by combining immunoaffinity purification and LC-MS/MS without depletion of abundant proteins. The method was used to survey serum samples of patients with acute myeloid leukemia (AML), breast cancer (BC), and nonsmall cell lung cancer (NSCLC). Peptides were identified from serum samples containing phosphorylation, acetylation, lysine methylation, and arginine methylation. Of the PTMs identified, lysine acetylation (AcK) and arginine mono-methylation (Rme) were more prevalent than other PTMs. Label-free quantitative analysis of AcK and Rme peptides was performed for sera from AML, BC, and NSCLC patients. Several AcK and Rme sites showed distinct abundance distribution patterns across the three cancer types. The identification and quantification of posttranslationally modified peptides in serum samples reported here can be used for patient profiling and biomarker discovery research. A robust method was developed and optimized for enrichment and quantitative analysis of posttranslational modifications (PTMs) in serum/plasma samples by combining immunoaffinity purification and LC-MS/MS without depletion of abundant proteins. The method was used to survey serum samples of patients with acute myeloid leukemia (AML), breast cancer (BC), and nonsmall cell lung cancer (NSCLC). Peptides were identified from serum samples containing phosphorylation, acetylation, lysine methylation, and arginine methylation. Of the PTMs identified, lysine acetylation (AcK) and arginine mono-methylation (Rme) were more prevalent than other PTMs. Label-free quantitative analysis of AcK and Rme peptides was performed for sera from AML, BC, and NSCLC patients. Several AcK and Rme sites showed distinct abundance distribution patterns across the three cancer types. The identification and quantification of posttranslationally modified peptides in serum samples reported here can be used for patient profiling and biomarker discovery research. Biomarker identification is a key step for illustration of disease mechanisms, drug development, and diagnostics. Diagnostics research has focused on identifying biomarkers from viable biofluids, including serum/plasma, saliva, cerebrospinal fluid, and urine. Due to ease of collection and richness in proteins and metabolites, serum/plasma has been the preferred choice for diagnostic studies (1.Galasko D. Golde T.E. Biomarkers for Alzheimer's disease in plasma, serum and blood—Conceptual and practical problems.Alzheimer's Res. Therapy. 2013; 5: 10Crossref PubMed Scopus (49) Google Scholar, 2.Ahn J.M. Sung H.J. Yoon Y.H. Kim B.G. Yang W.S. Lee C. Park H.M. Kim B.J. Kim B.G. Lee S.Y. An H.J. Cho J.Y. Integrated glycoproteomics demonstrates fucosylated serum paraoxonase 1 alterations in small cell lung cancer.Mol. Cell. Proteomics. 2014; 13: 30-48Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 3.Yotsukura S. Mamitsuka H. Evaluation of serum-based cancer biomarkers: A brief review from a clinical and computational viewpoint.Crit. Rev. Oncol. Hematol. 2015; 93: 103-115Crossref PubMed Scopus (38) Google Scholar). Advancement of mass-spectrometry-based proteomic technologies has allowed identification and quantification of thousands of proteins in serum/plasma samples. Typically, these methods combine isotopic labeling, offline fractionation, and LC-MS/MS analysis. Facilitated by high-throughput proteomics analysis, researchers have collected vast amounts of comparative information about protein abundance in serum/plasma of patients of various types of diseases that accelerated the identification of potential biomarkers (4.Keshishian H. Burgess M.W. Gillette M.A. Mertins P. Clauser K.R. Mani D.R. Kuhn E.W. Farrell L.A. Gerszten R.E. Carr S.A. Multiplexed, quantitative workflow for sensitive biomarker discovery in plasma yields novel candidates for early myocardial injury.Mol. Cell. Proteomics. 2015; 14: 2373-2393Abstract Full Text Full Text PDF Scopus (145) Google Scholar). To date, the majority of serum/plasma proteomic work has been conducted to analyze total protein level abundance, with only a few studies to analyze posttranslational modifications (PTMs) 1The abbreviations used are:PTMposttranslational modificationAMLacute myeloid leukemiaBCbreast cancerNSCLCnonsmall cell lung cancerAcKlysine acetylationRmearginine mono-methylationIMACimmobilized metal ion affinity chromatographyhnRNPheterogeneous nuclear ribonucleoprotein., usually glycosylation (5.Berven F.S. Ahmad R. Clauser K.R. Carr S.A. Optimizing performance of glycopeptide capture for plasma proteomics.J. Proteome Res. 2010; 9: 1706-1715Crossref PubMed Scopus (47) Google Scholar, 6.Chen S. Lu C. Gu H. Mehta A. Li J. Romano P.B. Horn D. Hooper D.C. Bazemore-Walker C.R. Block T. Aleuria aurantia lectin (AAL)-reactive immunoglobulin G rapidly appears in sera of animals following antigen exposure.PloS One. 2012; 7: e44422Crossref PubMed Scopus (13) Google Scholar). As one of the most important mechanisms for regulating protein function, PTMs, including phosphorylation, acetylation, ubiquitination, and methylation, have been identified and validated as critical for signaling transduction, protein degradation, and transcriptional regulation (7.Prabakaran S. Lippens G. Steen H. Gunawardena J. Post-translational modification: Nature's escape from genetic imprisonment and the basis for dynamic information encoding.Wiley Interdisciplinary Rev. Syst. Biol. Med. 2012; 4: 565-583Crossref PubMed Scopus (235) Google Scholar, 8.Huang H. Lin S. Garcia B.A. Zhao Y. Quantitative proteomic analysis of histone modifications.Chemical Rev. 2015; 115: 2376-2418Crossref PubMed Scopus (253) Google Scholar). Currently, there exists very limited data about PTMs in serum/plasma beyond glycosylation. The abundant serum protein albumin has long been known to be acetylated by aspirin, and this reaction can occur in vitro without the presence of any acetyltransferase (9.Liyasova M.S. Schopfer L.M. Lockridge O. Reaction of human albumin with aspirin in vitro: Mass spectrometric identification of acetylated lysines 199, 402, 519, and 545.Biochem. Pharmacol. 2010; 79: 784-791Crossref PubMed Scopus (65) Google Scholar). Fibrinogen, another abundant serum protein, is also acetylated by aspirin both in vivo and in vitro (10.Bjornsson T.D. Schneider D.E. Berger Jr., H. Aspirin acetylates fibrinogen and enhances fibrinolysis. Fibrinolytic effect is independent of changes in plasminogen activator levels.J. Pharmacol. Exp. Therapeut. 1989; 250: 154-161PubMed Google Scholar, 11.Pinckard R.N. Hawkins D. Farr R.S. In vitro acetylation of plasma proteins, enzymes and DNA by aspirin.Nature. 1968; 219: 68-69Crossref PubMed Scopus (156) Google Scholar). These previous findings and the known importance of PTMs in cellular signaling provided the impetus for a large-scale survey of PTMs other than glycosylation by immunoaffinity enrichment of PTM-containing peptides. posttranslational modification acute myeloid leukemia breast cancer nonsmall cell lung cancer lysine acetylation arginine mono-methylation immobilized metal ion affinity chromatography heterogeneous nuclear ribonucleoprotein. One challenge for proteomic analysis of serum/plasma is the broad dynamic range of the serum/plasma proteome (12.Mitchell P. Proteomics retrenches.Nature Biotech. 2010; 28: 665-670Crossref PubMed Scopus (81) Google Scholar), including a high percentage of the total protein content of serum/plasma represented by only 12 proteins. This limitation can be partially overcome by immunodepletion of abundant proteins prior to enzymatic digestion (4.Keshishian H. Burgess M.W. Gillette M.A. Mertins P. Clauser K.R. Mani D.R. Kuhn E.W. Farrell L.A. Gerszten R.E. Carr S.A. Multiplexed, quantitative workflow for sensitive biomarker discovery in plasma yields novel candidates for early myocardial injury.Mol. Cell. Proteomics. 2015; 14: 2373-2393Abstract Full Text Full Text PDF Scopus (145) Google Scholar, 13.Adkins J.N. Varnum S.M. Auberry K.J. Moore R.J. Angell N.H. Smith R.D. Springer D.L. Pounds J.G. Toward a human blood serum proteome: Analysis by multidimensional separation coupled with mass spectrometry.Mol. Cell. Proteomics. 2002; 1: 947-955Abstract Full Text Full Text PDF PubMed Scopus (719) Google Scholar), however, generation of the large quantities of materials necessary for PTM enrichment with an immunodepletion workflow could be cost prohibitive. It was therefore of interest to develop a PTM enrichment workflow from serum/plasma without the need for depletion of the abundant proteins. This method allows PTM profiling from a reasonable volume of serum (∼250 μl for multiple PTM enrichment) followed by LC-MS/MS analysis. Among the PTMs surveyed, lysine acetylation (AcK) and arginine mono-methylation (Rme) were identified as the more prevalent PTMs in cancer patients' sera. These PTMs were profiled in sera from patients with acute myelogenous leukemia (AML), breast cancer (BC), and nonsmall cell lung cancer (NSCLC). At 1% FDR, we have identified 796 unique AcK sites and 808 unique Rme sites in the sera of 12 cancer patients. The abundant serum protein human albumin was identified acetylated at 59 different sites, while other abundant proteins were also found to be acetylated, including A2M and serotransferrin. About 25% of the identified AcK sites (190 out of 796) were from the 12 most abundant serum proteins. In contrast, the Rme sites identified were from a more diverse complement of proteins, including transcriptional regulators and RNA processing proteins. Quantitative analysis identified a subset of peptides in each enrichment with differential abundance across the three cancer types surveyed. For example, the abundance of a Lys155-containing peptide from the complement component 3 protein was higher in the sera of NSCLC patients compared with AML and BC patients. Conversely, the abundance of an Arg1593 mono-methylated peptide from protein ARID1A was lower in the sera of NSCLC than the other two cancer types. Clustering of the quantitative data for the AcK and Rme enrichments revealed patterns of modification specific to cancer type as well as patient pathology. Together, these data demonstrate the utility of PTM profiling of human serum samples for disease characterization and the potential for biomarker discovery. Serum samples of four patients of AML, BC, and NSCLC were purchased from Proteogenex (Culver City, CA). Patient information and the concentration of total protein of each serum sample are provided in Supplemental Table S1. Serum samples were processed using the PTMScan method as previously described (14.Guo A. Gu H. Zhou J. Mulhern D. Wang Y. Lee K.A. Yang V. Aguiar M. Kornhauser J. Jia X. Ren J. Beausoleil S.A. Silva J.C. Vemulapalli V. Bedford M.T. Comb M.J. Immunoaffinity enrichment and mass spectrometry analysis of protein methylation.Mol. Cell. Proteomics. 2014; 13: 372-387Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). Equal volumes of serum (250 μl for individual sample) were mixed with urea lysis buffer (9 m sequanol-grade urea, 20 mm HEPES, pH 8.0, 1 mm β-glycerophosphate, 1 mm sodium vanadate, 2.5 mm sodium pyrophosphate) to a final concentration of 6 m urea. For the technical triplicate experiment, 150 μl serum from four nonsmall cell lung cancer patients were pooled together and split into three aliquots for independent processing. The samples were centrifuged at 16,000 × g for 15 min at 4 °C. Supernatants were collected and reduced with 4.5 mm DTT for 30 min at 55 °C. Reduced lysates were alkylated with iodoacetimide (0.095g per 5 ml H2O) for 15 min at room temperature in the dark. Samples were diluted 1:4 with 20 mm HEPES, pH 8.0, and digested overnight with 10 μg/ml trypsin-TPCK (Worthington, #LS003740, Lakewood, NJ). Digested peptide lysates were acidified with 1% TFA and peptides were desalted over 360 mg SEP PAK Classic C18 columns (Waters, #WAT051910, Milford, MA). Peptides were eluted with 40% acetonitrile in 0.1% TFA, dried under vacuum, and stored at −80 °C. Enrichment of posttranslationally modified peptides was performed using the antibodies listed in Table I. following protocols described previously (14.Guo A. Gu H. Zhou J. Mulhern D. Wang Y. Lee K.A. Yang V. Aguiar M. Kornhauser J. Jia X. Ren J. Beausoleil S.A. Silva J.C. Vemulapalli V. Bedford M.T. Comb M.J. Immunoaffinity enrichment and mass spectrometry analysis of protein methylation.Mol. Cell. Proteomics. 2014; 13: 372-387Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 15.Svinkina T. Gu H. Silva J.C. Mertins P. Qiao J. Fereshetian S. Jaffe J.D. Kuhn E. Udeshi N.D. Carr S.A. Deep, quantitative coverage of the lysine acetylome using novel anti-acetyl-lysine antibodies and an optimized proteomic workflow.Mol. Cell. Proteomics. 2015; 14: 2429-2440Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Briefly, saturating amounts of the indicated antibodies were bound to 30 μl packed Protein A agarose beads (Roche, Indianapolis, IN) overnight at 4 °C. Lyophilized serum peptides were resuspended in MOPS IAP buffer (50 mm MOPS, pH 7.2, 10 mm KH2PO4, 50 mm NaCl) and centrifuged 5 min at 10,000 × g. Supernatants were mixed with antibody bead slurries for 2 h at 4 °C. Beads were pelleted by centrifugation 30 s at 2,000 × g at 4 °C. Beads were washed three times with 1.5 ml IAP buffer containing 1% Nonidet P-40 and three times with 1 ml water (Burdick and Jackson, Morristown, NJ). Peptides were eluted from beads with 0.15% TFA (sequential elutions of 40 μl followed by 35 μl, 10 min each at room temperature). Eluted peptides were desalted over tips packed with Empore C18 (Sigma, St. Louis, MO) and eluted with 40% acetonitrile in 0.1% TFA. Eluted peptides were dried under vacuum and subject to a second in-solution trypsin digest using 250 ng of sequencing grade trypsin (Promega, Madison, WI) in 50 mm ammonium bicarbonate/5% acetonitrile for 2 h at 37 °C to minimize miscleavage and digest any antibody remaining in the sample. Samples were acidified with TFA and repurified over C18 tips as before.Table ISummary of posttranslationally modified peptides identified using various enrichments from pooled sera of cancer patientsExp. #SampleEnrichment MethodPTM typeRaw fileUnique modified peptide1AMLIMAC-Fe3+pSTY231681482BCIMAC-Fe3+pSTY231691843NSCLCIMAC-Fe3+pSTY231701454AMLpY motif antibodypY23231395BCpY motif antibodypY23232396NSCLCpY motif antibodypY23233447AMLpY → IMAC-Fe3+pY23234198BCpY → IMAC-Fe3+pY23235269NSCLCpY → IMAC-Fe3+pY232362510AMLAcK motif antibodyAcK2317135511BCAcK motif antibodyAcK2317235912NSCLCAcK motif antibodyAcK2317343113AMLRme motif antibodyRme2323820614BCRme motif antibodyRme2323917215NSCLCRme motif antibodyRme2324013816AMLPan-Kme antibodyKme (mono, di, tri)2324111717BCPan-Kme antibodyKme (mono, di, tri)2324214218NSCLCPan-Kme antibodyKme (mono, di, tri)23243148 Open table in a new tab IMAC enrichment was performed as previously described (16.Ficarro S.B. Zhang Y. Carrasco-Alfonso M.J. Garg B. Adelmant G. Webber J.T. Luckey C.J. Marto J.A. Online nanoflow multidimensional fractionation for high efficiency phosphopeptide analysis.Mol. Cell. Proteomics. 2011; 10 (O111.011064)Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Nickel-agarose magnetic beads (Qiagen, Valencia, CA) were treated with EDTA to remove the nickel, washed three times with H2O, loaded with aqueous FeCl3 for 30 min, and washed. For phosphopeptide enrichment, 10 μl Fe3+-agarose slurry were added to peptide digested from 10 μl of serum in 1 ml 0.1% TFA/80% acetonitrile for 30 min at room temperature. Unbound peptides were removed by washing three times with 0.1% TFA/80% MeCN. Bound peptides were eluted with 2X 50 μl of 2.5% ammonia/50% acetonitrile solution for 5 min. The eluent was immediately acidified by 20% TFA and dried in a speed-vac. Samples were resuspended in 50 μl 0.15% TFA, desalted over C18 tips, and redried in a speed vac. Immunoprecipitated peptides were resuspended in 0.125% formic acid and separated on a reversed-phase C18 column (75 μm inner diameter × 10 cm) packed into a PicoTip emitter (∼8 μm inner diameter) with Magic C18 AQ (100Å × 5 μm, New Objective, Woburn, MA). Each sample was split, and analytical replicate injections were run to increase the number of identifications and provide metrics for analytical reproducibility of the method. A standard peptide mix (MassPREPTM Protein Digestion Standard Mix 1, Waters) was spiked in each sample vial in a total quantity of 100 fmol (33 fmol per injection) prior to LC-MS/MS analysis. For antibody enrichment, peptides from 120 μl serum were run per injection; for IMAC, peptides from 5 μl serum were run per injection. Replicate injections were run nonsequentially to reduce artificial changes in peptide abundance due to changes in instrument performance over time. One replicate of each sample was injected then the second replicate in reverse order. Peptides were eluted using a 120-min or 150-min linear gradient of acetonitrile in 0.125% formic acid delivered at 280 nl/min from 3% to 30% acetonitrile. Tandem mass spectra were collected in a data-dependent manner with an LTQ-Orbitrap ELITE mass spectrometer (Thermo Fisher Scientific, Waltham, MA) running XCalibur 2.0.7 SP1 using a top-20 MS/MS method, a dynamic repeat count of one, and a repeat duration of 30 s. The isolation window was set at 1.0 Da with a normalized collision energy of 35%. Real-time recalibration of mass error was performed using lock mass (17.Olsen J.V. de Godoy L.M. Li G. Macek B. Mortensen P. Pesch R. Makarov A. Lange O. Horning S. Mann M. Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap.Mol. Cell. Proteomics. 2005; 4: 2010-2021Abstract Full Text Full Text PDF PubMed Scopus (1243) Google Scholar) with a singly charged polysiloxane ion m/z = 371.101237. The data associated with this manuscript including labeled MS2 spectra in Skyline library format may be downloaded from the ProteomeXchange Consortium via PRIDE with project accession numbers: PXD002931 [username: [email protected]; password: wpPsK3wN] and PXD002932 [username: [email protected]; password: qT4I4DrS]. MS/MS spectra were evaluated using SEQUEST and the Core platform from Harvard University (18.Eng J.K. McCormack A.L. Yates J.R. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database.J. Am. Soc. Mass Spectr. 1994; 5: 976-989Crossref PubMed Scopus (5434) Google Scholar, 19.Huttlin E.L. Jedrychowski M.P. Elias J.E. Goswami T. Rad R. Beausoleil S.A. Villén J. Haas W. Sowa M.E. Gygi S.P. A tissue-specific atlas of mouse protein phosphorylation and expression.Cell. 2010; 143: 1174-1189Abstract Full Text Full Text PDF PubMed Scopus (1217) Google Scholar, 20.Villén J. Beausoleil S.A. Gerber S.A. Gygi S.P. Large-scale phosphorylation analysis of mouse liver.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 1488-1493Crossref PubMed Scopus (627) Google Scholar). Files were searched against the NCBI homo sapiens FASTA database updated on June 27, 2011 containing 34,899 forward and 34,899 reverse sequences. A mass accuracy of ±5 ppm was used for precursor ions and 1 Da for product ions. Enzyme specificity was limited to trypsin, with at least one tryptic (K- or R-containing) terminus required per peptide and up to four miscleavages allowed. Cysteine carboxamidomethylation was specified as a static modification; oxidation of methionine residue and the appropriate PTM were allowed as variable modifications for each enrichment sample set. Reverse decoy databases were included for all searches to estimate false discovery rates, and filtered using a 1% FDR in the Linear Discriminant module of Core. All quantitative results were generated using Progenesis V4.1 (Waters Cooperation) and Skyline Version 3.1 to extract the integrated peak area of the corresponding peptide assignments according to previously published protocols (21.Schilling B. Rardin M.J. MacLean B.X. Zawadzka A.M. Frewen B.E. Cusack M.P. Sorensen D.J. Bereman M.S. Jing E. Wu C.C. Verdin E. Kahn C.R. Maccoss M.J. Gibson B.W. Platform-independent and label-free quantitation of proteomic data using MS1 extracted ion chromatograms in skyline: Application to protein acetylation and phosphorylation.Mol. Cell. Proteomics. 2012; 11: 202-214Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 22.Gnad F. Young A. Zhou W. Lyle K. Ong C.C. Stokes M.P. Silva J.C. Belvin M. Friedman L.S. Koeppen H. Minden A. Hoeflich K.P. Systems-wide analysis of K-Ras, Cdc42, and PAK4 signaling by quantitative phosphoproteomics.Mol. Cell. Proteomics. 2013; 12: 2070-2080Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Extracted ion chromatograms for peptide ions that changed in abundance between samples were manually reviewed to ensure accurate quantitation in Skyline. Statistical analysis of the quantitative data was done using a two-tailed t test between two cancer groups. The maximum negative log-p value from three comparison pairs was used to indicate significance for abundance changes of a certain peptide between two cancer groups. False discovery rate for each binary comparison was further controlled by applying the Benjamini–Hochberg procedure. Heat maps of the quantitative data were generated and clustered in Spotfire DecisionSite (TIBCO Software AB) version 9.1.2. Equal volumes of serum samples were mixed with SDS-PAGE sample buffer (Cell Signaling Technology, Danvers, MA #7723,) and run on 4–20% gradient tris-glycine gels (Invitrogen Grand Island, NY). For pan-AcK and pan-Rme Western blots, serum was diluted 10-fold and 20 μl were loaded. For albumin, serum was diluted 10,000- fold and 20 μl were loaded. Proteins were transferred to nitrocellulose (Millipore, Bedford, MA) and blocked for 1 h in 5% nonfat dry milk (Sigma) in TBS. Primary antibodies (pan-AcK (#13420), pan-Rme (#8015/#8711), and albumin (#4929)) were incubated in 5% BSA in TBS plus 0.1% Tween-20 overnight at 4 °C. Membranes were washed three times with TBS plus 0.1% Tween-20, incubated with anti-rabbit secondary antibody (#5366) for 1 h at room temperature in 5% milk and TBS plus 0.1% Tween-20, washed three times with TBS plus 0.1% Tween-20, dried, and developed on the Odyssey near-infrared imaging system (LI-COR). All antibodies were from Cell Signaling Technology. To demonstrate an optimal workflow for analysis of posttranslational modifications in serum samples, we first tested enrichment using various PTM antibodies and IMAC-Fe3+ with pooled serum samples as outlined in Table I. Serum samples were directly processed for PTM enrichment without any prior depletion of abundant serum proteins. The enrichments performed included IMAC-Fe3+, phosphotyrosine enrichment, phosphotyrosine enrichment followed by IMAC-Fe3+, acetyl-lysine enrichment (AcK), arginine mono-methylation (Rme), and lysine pan-methylation. Of these enrichments, acetyl-lysine and mono-methyl-arginine showed the most promising results, with the highest number of peptides per sample (Table I). The number of identifications for the phospho-enrichment was low and failed to identify proteins/sites known to be important disease drivers or substrates for the cancer types profiled (data not shown). Having established the ability to profile PTMs directly from serum samples, we then applied the immunoaffinity enrichment method to profile lysine acetylation and arginine mono-methylation in patient sera of acute myeloid leukemia (AML), breast cancer (BC), and nonsmall cell lung cancer (NSCLC), respectively (n = 4 for each type of cancer, 12 samples total). As different PTMs were being profiled, PTM enrichment was performed sequentially on the same sample as previously described (23.Mertins P. Qiao J.W. Patel J. Udeshi N.D. Clauser K.R. Mani D.R. Burgess M.W. Gillette M.A. Jaffe J.D. Carr S.A. Integrated proteomic analysis of post-translational modifications by serial enrichment.Nat. Methods. 2013; 10: 634-637Crossref PubMed Scopus (434) Google Scholar). To minimize interference from factors such as sex, age, and ethnic background, the study included only female patients of Caucasian background (Supplemental Table S1). The general workflow of the sequential enrichment is outlined in Fig. 1. Prior to immunoaffinity enrichment, Western blotting using pan-AcK and Rme motif antibodies were performed on the 12 patient samples (Supplemental Fig. S1). Most of the AcK signal in the Western blotting fell into a molecular weight range between 58–80 kDa, corresponding to human albumin (MW = 69kDa), with other lower intensity bands visible throughout the MW range. In the Rme blot, the strong signal from albumin was not observed, with other bands detected at various molecular weights. Enrichment for AcK generated a range of 214 to 486 unique AcK peptides from each serum sample, while enrichment for Rme generated a range of 199 to 257 unique Rme peptides from each serum sample (Supplemental Tables S2 and S3, Details Tab). Many AcK and Rme sites identified were represented by multiple peptides due to methionine oxidation, miscleavage, and different charge states. Tables were made nonredundant by unique protein site (Supplemental Tables S2 and S3, Summary Tab) using the peptide with the largest number of MS/MS identifications (Count in Details Column, Summary Tab, Supplemental Table S2 and S3) as the best representative for a particular PTM site. In total, 796 and 808 unique sites were identified for AcK and Rme, respectively. Of these, 672 AcK sites and 619 Rme sites were previously unidentified and will be curated into the PhosphoSitePlus database as a public resource (24.Hornbeck P.V. Chabra I. Kornhauser J.M. Skrzypek E. Zhang B. PhosphoSite: A bioinformatics resource dedicated to physiological protein phosphorylation.Proteomics. 2004; 4: 1551-1561Crossref PubMed Scopus (444) Google Scholar). This study identified 520 and 688 unique proteins from AcK and Rme enrichment, respectively (Supplemental Table S4). The top 10 protein classes represented in each enrichment are shown in the pie charts in Figs. 2A (AcK) and 2B (Rme). For AcK sites, the top five protein categories were secreted protein, receptor/channel/transporter/cell surface protein, adhesion/extracellular protein, chromatin/DNA-binding/DNA repair/DNA replication protein, and transcriptional regulator. Peptides from albumin were identified acetylated at a total of 59 unique AcK sites, which is consistent with the Western blotting signal shown in Supplemental Fig. S1A. High numbers of acetylation sites were also identified on other serum-abundant proteins, including alpha-2-macroglobulin and serotransferrin with a total number of 35 and 29 unique AcK sites identified, respectively. Overall, about 25% of all AcK sites identified (190 out of 796) were from the top 12 abundant serum proteins. Conversely, for Rme, only 4 out of 808 unique Rme sites from top 12 abundant serum proteins were identified. The top five protein categories for Rme were receptor/channel/transporter/cell surface protein, RNA processing, transcriptional regulator, adhesion/extracellular protein, and adaptor/scaffold. A large number of unique Rme sites was identified from various heterogeneous nuclear ribonucleoprotein (hnRNP) isoforms, most of which have been identified before in a previous study (14.Guo A. Gu H. Zhou J. Mulhern D. Wang Y. Lee K.A. Yang V. Aguiar M. Kornhauser J. Jia X. Ren J. Beausoleil S.A. Silva J.C. Vemulapalli V. Bedford M.T. Comb M.J. Immunoaffinity enrichment and mass spectrometry analysis of protein methylation.Mol. Cell. Proteomics. 2014; 13: 372-387Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). The presence of these posttranslationally modified hnRNP peptides in serum/plasma has not been previously reported. The data for the two enrichments were largely complementary, with only 35 proteins identified in common (Supplemental Table S4, Supplemental Fig. S2). There was also a low degree of overlap between these results and a recent large-scale plasma proteome study using iTRAQ labeling and offline fractionation prior to LC-MS/MS analysis, which identified over 5300 proteins with high confidence (4.Keshishian H. Burgess M.W. Gillette M.A. Mertins P. Clauser K.R. Mani D.R. Kuhn E.W. Farrell L.A. Gerszten R.E. Carr S.A. Multiplexed, quantitative workflow for sensitive biomarker discovery in plasma yields novel candidates for early myocardial injury.Mol. Cell. Proteomics. 2015; 14: 2373-2393Abstract Full Text Full Text PDF Scopus (145) Google Scholar), with 226 out of 520 proteins in common for AcK and 212 out of 688 for Rme (Supplemental Fig. S2). A pooled mixture of NSCLC patient serum was split into three aliquots and subject to parallel, independent trypsin digestion, sequential immuno-enrichment for AcK and Rme, and LC-MS/MS analysis. We have identified 555, 502, and 515 unique AcK peptides and 373, 357, and 377 unique Rme peptides from the three independent samples, respectively. A total of 778 unique AcK peptides and 564 unique Rme peptides were identified across the triplicate runs, of which, 361 (46%) AcK peptide identifications and 226 (40%) Rme peptide identifications were shared by all three samples (Supplemental Fig. S3). Of the unique peptides identified, 380 AcK and 356 Rme were quantified using the label-free quantification approach (Supplemental Tables S5 and S6). The distributions of %CV between technical triplicates for the two PTMs are shown in Supplemental Fig. S4. The median %CVs were 23 and 17% for lysine acetylation and arginine mono-methylation, respectively. About 78% of acetyl-lysine peptides and 94% of mono methyl-arginine pep