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Isotope-labeled Protein Standards

2007; Elsevier BV; Volume: 6; Issue: 12 Linguagem: Inglês

10.1074/mcp.m700163-mcp200

1535-9484

Virginie Brun, Alain Dupuis, Annie Adrait, Marlène Marcellin, Damien Thomas, Magali Court, François Vandenesch, Jérôme Garin,

Biosensors and Analytical Detection

Diagnostic development and public health surveillance require technologies that provide specific identification and absolute quantification of protein biomarkers. Beside immunologically related techniques (e.g. enzyme-linked immunosorbent assay), MS is gaining increasing interest due to its high sensitivity and specificity. Furthermore, MS-based analyses are extremely accurate quantitatively, provided that suitable reference standards are available. Recently, the use of chemically synthesized isotope-labeled marker peptides for MS-based absolute quantification of proteins has led to major advances. However, we show here that the use of such peptides can lead to severe biases. In this work, we present an innovative strategy (Protein Standard Absolute Quantification) that uses in vitro-synthesized isotope-labeled full-length proteins as standards for absolute quantification. As those protein standards perfectly match the biochemical properties of the target proteins, they can be directly added into the samples to be analyzed, allowing a highly accurate quantification of proteins even in prefractionated complex samples. The power of our Protein Standard Absolute Quantification methodology for accurate absolute quantification of biomarkers was demonstrated both on water and urine samples contaminated with Staphylococcus aureus superantigenic toxins as typical biomarkers of public health interest. Diagnostic development and public health surveillance require technologies that provide specific identification and absolute quantification of protein biomarkers. Beside immunologically related techniques (e.g. enzyme-linked immunosorbent assay), MS is gaining increasing interest due to its high sensitivity and specificity. Furthermore, MS-based analyses are extremely accurate quantitatively, provided that suitable reference standards are available. Recently, the use of chemically synthesized isotope-labeled marker peptides for MS-based absolute quantification of proteins has led to major advances. However, we show here that the use of such peptides can lead to severe biases. In this work, we present an innovative strategy (Protein Standard Absolute Quantification) that uses in vitro-synthesized isotope-labeled full-length proteins as standards for absolute quantification. As those protein standards perfectly match the biochemical properties of the target proteins, they can be directly added into the samples to be analyzed, allowing a highly accurate quantification of proteins even in prefractionated complex samples. The power of our Protein Standard Absolute Quantification methodology for accurate absolute quantification of biomarkers was demonstrated both on water and urine samples contaminated with Staphylococcus aureus superantigenic toxins as typical biomarkers of public health interest. Mass spectrometry MS has greatly contributed to the maturation of proteomics (1Aebersold R. Constellations in a cellular universe.Nature. 2003; 422: 115-116Crossref PubMed Scopus (78) Google Scholar). It is now possible to characterize hundreds of proteins in an hour time frame and compare protein abundances in pairs of samples. The next frontier lies in accurate absolute quantitation. Although label-free spectral counting approaches (2Lu P. Vogel C. Wang R. Yao X. Marcotte E.M. Absolute protein expression profiling estimates the relative contributions of transcriptional and translational regulation.Nat. Biotechnol. 2007; 25: 117-124Crossref PubMed Scopus (916) Google Scholar, 3Mallick P. Schirle M. Chen S.S. Flory M.R. Lee H. Martin D. Ranish J. Raught B. Schmitt R. Werner T. Kuster B. Aebersold R. Computational prediction of proteotypic peptides for quantitative proteomics.Nat. Biotechnol. 2007; 25: 125-131Crossref PubMed Scopus (572) Google Scholar) are attracting considerable interest, robust absolute quantitative methodologies typically rely on the well-established isotope dilution principle (4Tai S.S. Bunk D.M. White E.T. Welch M.J. Development and evaluation of a reference measurement procedure for the determination of total 3,3′,5-triiodothyronine in human serum using isotope-dilution liquid chromatography-tandem mass spectrometry.Anal. Chem. 2004; 76: 5092-5096Crossref PubMed Scopus (72) Google Scholar), in which internal standardization is achieved with isotope-labeled homologs of specific proteolytic peptides from the target protein(s) (5Gerber 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, 6Kirkpatrick 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). The Absolute Quantitation (AQUA) 1The abbreviations used are: AQUA, Absolute Quantification; AAA, amino acid analysis; MRM, multiple reaction monitoring; PSAQ, protein standard absolute quantification; QconCAT, concatemer of standard peptides for absolute quantification; SEA, staphylococcal enterotoxin A; SEB, staphylococcal enterotoxin B; TSST-1, toxic shock syndrome toxin-1. peptide strategy uses chemically synthesized isotope-labeled peptides which are spiked into the samples in known quantities before MS analysis (5Gerber 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, 6Kirkpatrick 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, 7Stemmann O. Zou H. Gerber S.A. Gygi S.P. Kirschner M.W. Dual inhibition of sister chromatid separation at metaphase.Cell. 2001; 107: 715-726Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar, 8Barr J.R. Maggio V.L. Patterson Jr., D.G. Cooper G.R. Henderson L.O. Turner W.E. Smith S.J. Hannon W.H. Needham L.L. Sampson E.J. Isotope dilution–mass spectrometric quantification of specific proteins: model application with apolipoprotein A-I.Clin. Chem. 1996; 42: 1676-1682Crossref PubMed Scopus (321) Google Scholar). Recently, the synthesis and metabolic labeling of an artificial concatemer of standard peptides (QconCAT), which can be spiked into the samples before trypsin digestion, was introduced to extend the number of quantified proteins (9Beynon R.J. Doherty M.K. Pratt J.M. Gaskell S.J. Multiplexed absolute quantification in proteomics using artificial QCAT proteins of concatenated signature peptides.Nat. Methods. 2005; 2: 587-589Crossref PubMed Scopus (395) Google Scholar, 10Anderson 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, 11Pratt J.M. Simpson D.M. Doherty M.K. Rivers J. Gaskell S.J. Beynon R.J. Multiplexed absolute quantification for proteomics using concatenated signature peptides encoded by QconCAT genes.Nat. Protoc. 2006; 1: 1029-1043Crossref PubMed Scopus (305) Google Scholar, 12Rivers J. Simpson D.M. Robertson D.H.L. Gaskell S.J. Beynon R.J. Absolute multiplexed quantitative analysis of protein expression during muscle development using QconCAT.Mol. Cell. Proteomics. 2007; 6: 1416-1427Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Although AQUA and QconCAT approaches have significantly advanced the quantitative measurement of proteins in biological samples, calibration with AQUA peptides and QconCAT constructs suffer from the following limitations: 1) a failure to take into account the actual efficiency of the proteolysis step required before MS analysis; 2) an incompatibility with sample prefractionation, which is often necessary when dealing with biological samples (13Shen Y. Kim J. Strittmatter E.F. Jacobs J.M. Camp 2nd, D.G. Fang R. Tolie N. Moore R.J. Smith R.D. Characterization of the human blood plasma proteome.Proteomics. 2005; 5: 4034-4045Crossref PubMed Scopus (176) Google Scholar); and 3Mallick P. Schirle M. Chen S.S. Flory M.R. Lee H. Martin D. Ranish J. Raught B. Schmitt R. Werner T. Kuster B. Aebersold R. Computational prediction of proteotypic peptides for quantitative proteomics.Nat. Biotechnol. 2007; 25: 125-131Crossref PubMed Scopus (572) Google Scholar) a poor protein sequence coverage, limiting the statistical reliability of the quantification. We propose here an original Protein Standard Absolute Quantification (PSAQ) strategy for the absolute quantification of trace proteins in complex samples. This strategy is based on the use of in vitro-synthesized isotope-labeled full-length proteins as standards for quantification. To validate our methodology, we compared it with the two alternatives, AQUA and QconCAT strategies, using Staphylococcus aureus superantigenic toxins in water and urine samples as typical biomarkers of public health concern (14McCormick J.K. Yarwood J.M. Schlievert P.M. Toxic shock syndrome and bacterial superantigens: an update.Annu. Rev. Microbiol. 2001; 55: 77-104Crossref PubMed Scopus (577) Google Scholar, 15Dinges M.M. Orwin P.M. Schlievert P.M. Exotoxins of Staphylococcus aureus.Clin. Microbiol. Rev. 2000; 13: 16-34Crossref PubMed Scopus (1315) Google Scholar). S. aureus superantigenic toxins (enterotoxins and toxic shock syndrome toxin-1 [TSST-1]) are virulence factors responsible for severe diseases in humans among which the highly lethal staphylococcal toxic shock syndrome (14McCormick J.K. Yarwood J.M. Schlievert P.M. Toxic shock syndrome and bacterial superantigens: an update.Annu. Rev. Microbiol. 2001; 55: 77-104Crossref PubMed Scopus (577) Google Scholar, 15Dinges M.M. Orwin P.M. Schlievert P.M. Exotoxins of Staphylococcus aureus.Clin. Microbiol. Rev. 2000; 13: 16-34Crossref PubMed Scopus (1315) Google Scholar). Staphylococcal toxic shock syndrome is related to the ability of enterotoxins and TSST-1 to polyclonally and extensively activate T cells at picomolar concentrations, a property referred to as superantigenicity. Besides their superantigenic activity, staphylococcal enterotoxins also display specific emetic properties and constitute a major cause of food poisoning (15Dinges M.M. Orwin P.M. Schlievert P.M. Exotoxins of Staphylococcus aureus.Clin. Microbiol. Rev. 2000; 13: 16-34Crossref PubMed Scopus (1315) Google Scholar). For all these reasons, staphylococcal enterotoxins constitute a significant threat as biological weapons. The Center for Disease Control has registered the staphylococcal enterotoxin B (SEB) as a potential warfare contaminant of food and water supplies. To date, 19 staphylococcal enterotoxins have been identified (variant forms excluded) and are associated with different clinical profiles (15Dinges M.M. Orwin P.M. Schlievert P.M. Exotoxins of Staphylococcus aureus.Clin. Microbiol. Rev. 2000; 13: 16-34Crossref PubMed Scopus (1315) Google Scholar, 16Ferry T. Thomas D. Genestier A.L. Bes M. Lina G. Vandenesch F. Etienne J. Comparative prevalence of superantigen genes in Staphylococcus aureus isolates causing sepsis with and without septic shock.Clin. Infect. Dis. 2005; 41: 771-777Crossref PubMed Scopus (121) Google Scholar, 17Thomas D. Chou S. Dauwalder O. Lina G. Diversity in Staphylococcus aureus Enterotoxins.Chem. Immunol. Allergy. 2007; 93: 24-41Crossref PubMed Google Scholar). Extensive sequence and structure similarities between staphylococcal superantigenic toxins have so far precluded the development of specific and comprehensive immunological tools. As a result, no diagnostic test is presently available for the staphylococcal toxic shock syndrome. Furthermore, no assay is referenced for the identification and quantification of these toxins in accidentally or deliberately contaminated food or water supplies. In this context, MS, which circumvents the need for antibodies, offers great potential for staphylococcal superantigenic toxin-specific detection and quantification. In this work, we present an MS-based methodology to detect and quantify the staphylococcal superantigenic toxins SEA and TSST-1, which are the most frequently involved toxins in staphylococcal toxic shock syndrome (15Dinges M.M. Orwin P.M. Schlievert P.M. Exotoxins of Staphylococcus aureus.Clin. Microbiol. Rev. 2000; 13: 16-34Crossref PubMed Scopus (1315) Google Scholar, 16Ferry T. Thomas D. Genestier A.L. Bes M. Lina G. Vandenesch F. Etienne J. Comparative prevalence of superantigen genes in Staphylococcus aureus isolates causing sepsis with and without septic shock.Clin. Infect. Dis. 2005; 41: 771-777Crossref PubMed Scopus (121) Google Scholar). Drinking water and human urine were selected as preferred matrices for warfare agent surveillance and clinical diagnosis. Comparisons with the current state-of-the-art methodologies demonstrate the great potential of isotope-labeled full-length proteins as standards for MS-based absolute quantification. AQUA [13C6, 15N] l-leucine-labeled peptides were synthesized by Sigma-Genosys (Saint Quentin Fallavier, France). These peptides were quantified by amino acid analysis (AAA) by the provider. Recombinant staphylococcal enterotoxins SEA and TSST-1 were purchased from Toxin Technology (Sarasota, FL). The dilutions of quantification standards and commercial toxins were systematically performed in low adsorption tubes (Dutscher, Brumath, France). The QconCAT protein was designed as shown in Fig. 1. Briefly, tryptic peptides from eight staphylococcal superantigenic toxins (SEA, SEB, TSST-1, staphylococcal enterotoxin G, staphylococcal enterotoxin I, staphylococcal enterotoxin M, staphylococcal enterotoxin N, and staphylococcal enterotoxin O) were selected according to their uniqueness of sequence among the staphylococcal superantigens and their detectability in nano-LC-MS analysis. These peptide sequences were concatened into an artificial QconCAT protein and retro-translated to design the corresponding artificial QconCAT gene (see references 9Beynon R.J. Doherty M.K. Pratt J.M. Gaskell S.J. Multiplexed absolute quantification in proteomics using artificial QCAT proteins of concatenated signature peptides.Nat. Methods. 2005; 2: 587-589Crossref PubMed Scopus (395) Google Scholar and 11Pratt J.M. Simpson D.M. Doherty M.K. Rivers J. Gaskell S.J. Beynon R.J. Multiplexed absolute quantification for proteomics using concatenated signature peptides encoded by QconCAT genes.Nat. Protoc. 2006; 1: 1029-1043Crossref PubMed Scopus (305) Google Scholar for more details). The QconCAT gene was synthesized from 53 5′ phosphorylated oligonucleotides (Sigma-Genosys) covering the forward and reverse strands (Supplemental Fig. 1). The synthetic QconCAT gene was assembled by ligase chain reaction with TaqDNA ligase (New England Biolabs, Frankfurt, Germany) and amplified with the Expand High Fidelity polymerase (Roche, Meylan, France) using the primers mentioned in Supplemental Table I. The amplified QconCAT gene was purified, digested with NcoI (Roche) and SmaI (New England Biolabs), and inserted into the pIVEX 2.3d vector (Roche) providing a C-terminal hexahistidine purification tag. Ligation was achieved using the Rapid DNA Ligation Kit (Roche). The resulting plasmid was cloned into strain XL1-Blue (Stratagene, Amsterdam, Nederlands) and was purified using QIAprep Spin Miniprep Kit (Qiagen, Courtaboeuf, France). Finally, we checked the QconCAT construct sequence before its use for recombinant protein synthesis (Genome Express, Meylan, France). QconCAT protein production was performed in vitro using the RTS 500 ProteoMaster Escherichia coli HY Kit (Roche) according to the manufacturer's instructions with the following modifications: we used the RTS Amino Acid Sampler Kit (Roche) instead of the amino acid mix provided, and we replaced l-lysine and l-arginine by isotope-labeled [13C6, 15N2] l-lysine and [13C6, 15N4] l-arginine (Cambridge Isotope Laboratories, Andover, MA). The isotope enrichment of [13C6, 15N2] l-lysine and [13C6, 15N4] l-arginine was 98% 13C and 98% 15N. QconCAT protein was efficiently produced in a precipitated form and was solubilized in guanidine 6N. QconCAT purification was performed on a nickel affinity column (Ni Sepharose 6 Fast Flow, Amersham Biosciences, Freiburg, Germany) using a 20 mm-250 mm imidazole gradient in guanidine 6N. After purification, QconCAT was sequentially dialyzed against pure water and 1% SDS, Tris HCl 50 mm, pH 7.5. QconCAT quantification was performed by AAA on a Biochrom 30 Amino Acid Analyser (Biochrom, Cambridge, UK). QconCAT primary structure and isotope labeling was further assessed by nano-LC-MS/MS and nano-LC-MS analysis (Supplemental Fig. 2). Two S. aureus strains carrying SEA or TSST-1 gene were selected from the strain collection of the French National Staphylococci Reference Center. Isotope-labeled SEB standard was not synthesized as its production is officially restricted. Genomic DNA was prepared using the QIAamp DNA Stool Mini Kit (Qiagen). The primers used for PCR amplification are described in Supplemental Table I. SEA and TSST-1 PCR fragments were purified, digested with KspI (Roche) and SmaI (New England Biolabs), and cloned into the pIVEX 2.4d expression vector providing an N-terminal cleavable hexahistidine purification tag (Roche). Our PSAQ strategy relies on biochemical equivalence between each toxin and its PSAQ standard. We thus privileged an N terminus cleavable tag to allow a polishing of the limited N terminus heterogeneity reported for proteins produced by cell-free synthesis (18Torizawa T. Shimizu M. Taoka M. Miyano H. Kainosho M. Efficient production of isotopically labeled proteins by cell-free synthesis: a practical protocol.J. Biomol. NMR. 2004; 30: 311-325Crossref PubMed Scopus (106) Google Scholar). These constructs were cloned in Xl1 blue, purified, sequenced, and used for in vitro protein synthesis in the presence of [13C6, 15N2] l-lysine and [13C6, 15N4] l-arginine as described above for QconCAT. Isotope-labeled SEA and TSST-1 were readily produced in a soluble form and were purified on a nickel affinity column (Ni Sepharose 6 Fast Flow, Amersham Biosciences) using an imidazole gradient. The N-terminal hexahistidine tag of each isotope-labeled protein was cleaved by biotinylated Factor Xa (Factor Xa Removal Kit, Roche) according to the manufacturer's instructions. Both the resulting hexahistidine tag peptide and the biotinylated Factor Xa were removed in a single step using a mix of streptavidin coated beads and Ni Sepharose 6 Fast Flow resin. These isotope-labeled SEA and TSST-1 were quantified by AAA. Primary structure of the proteins and labeling efficiency were verified by nano-LC-MS/MS and nano-LC-MS analysis. The incorporation yield of [13C6, 15N2]-lysine and [13C6, 15N4]-arginine in these cell-free expressed standards was greater than 98% (data not shown). Home-produced PSAQ proteins and purchased SEA and TSST-1 were all checked for purity on SDS-PAGE using both Imperial Protein Stain and SYPRO Ruby staining (Bio-Rad, Marnes-la-Coquette, France). The quantities of commercial toxins were too limited for AAA analysis, and commercial TSST-1 toxin displayed a contamination by a higher molecular weight protein precluding an accurate AAA quantification. Thus, commercial toxin concentrations were evaluated by comparison with our AAA calibrated PSAQ standards on SDS-PAGE using SYPRO Ruby staining (19Nishihara J.C. Champion K.M. Quantitative evaluation of proteins in one- and two-dimensional polyacrylamide gels using a fluorescent stain.Electrophoresis. 2002; 23: 2203-2215Crossref PubMed Scopus (139) Google Scholar). SYPRO Ruby fluorescence was scanned (Laser 532 nm, filter 610BP30) on a Typhoon 9400 (Amersham Biosciences). When compared with our AAA calibrated PSAQ standards, the announced concentrations of commercial SEA and TSST toxins turned out to be overestimated (Supplemental Fig. 3). Therefore, the commercial toxin concentrations were corrected, and the corrected values were used in all subsequent calculations. Drinking water samples were contaminated with five different quantities of SEA and TSST-1 commercial toxins. Each sample was divided into nine aliquots of 120 μl each. Three aliquots (analytical replicates) of each sample were spiked with either QconCAT or PSAQ toxins standards in defined quantities. Trypsin digestion was performed in solution using sequencing grade modified trypsin (Promega, Madison, WI) at a 1:2 protease to toxins ratio in 25 mm NH4HCO3 overnight at 37 °C. Samples were dried by vacuum centrifugation and resolubilized in 5% ACN, 0.2% formic acid. Before nano-LC-MS analysis, AQUA peptides were added in defined quantities into the aliquots that contained neither QconCAT nor PSAQ standards. Urine from a 30-year-old healthy woman was collected and contaminated with four different quantities of SEA and TSST-1 commercial toxins. Each sample was divided into nine aliquots of 100 μl each. Three aliquots (analytical replicates) of each sample were contaminated with PSAQ toxin standards in defined amounts. Each 100 μl aliquot was adsorbed on 5 μl of Strataclean resin (Stratagene) according to the manufacturer's instructions. Following elimination of supernatant, proteins adsorbed onto the resin were directly eluted in 10 μl of a depolymerization buffer containing 2% SDS and 5% β-mercaptoethanol. At this stage, QconCAT standard was added in controlled quantities into half of the samples devoid of PSAQ standards. After a thermal denaturation step at 95 °C for 5 min, samples were loaded on precast Novex NuPAGE Bis–Tris gels (4–12% acrylamide gradient) purchased from Invitrogen (Cergy Pontoise, France). Gels were run for 30 min under 200 V, fixed for 30 min in 30% ethanol-7.5% acetic acid, and stained with Biosafe Coomassie blue (Bio-Rad). In the 25 kDa region of the gel encompassing toxins and QconCAT, protein bands were excised and were destained by repeated cycles of incubation in 25 mm NH4HCO3 for 15 min and then with 50% (v/v) ACN in the same buffer (25 mm NH4HCO3) for 15 min. After drying by vacuum centrifugation, the gel pieces were incubated with an oxidizing solution (7% H2O2) for 15 min (20Jaquinod M. Villiers F. Kieffer-Jaquinod S. Hugouvieux V. Bruley C. Garin J. Bourguignon J. A proteomics dissection of Arabidopsis thaliana vacuoles isolated from cell culture.Mol. Cell. Proteomics. 2007; 6: 394-412Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Gel pieces were then washed in HPLC grade water (Sigma-Aldrich) for 15 min before being dehydrated with 100% ACN. In-gel digestion was performed using 1:2 trypsin to protein ratio (sequencing grade modified trypsin, Promega) in 25 mm NH4HCO3 overnight at 37 °C. Peptides were extracted from the gel using passive diffusion in the following solutions: 50% ACN, then 5% formic acid, and finally 100% ACN. The extracts were dried by vacuum centrifugation, and peptides were resolubilized in 5% ACN, 0.2% formic acid. Before nano-LC-MS analysis, controlled amounts of AQUA peptides were added to the samples that had not been spiked with PSAQ or QconCAT standards. MS analyses were performed on a nano-LC system coupled to a QTOF Ultima mass spectrometer (Waters, Milford, MA). Briefly, peptide digests were first concentrated on a 300 μm × 5 mm PepMap C18 precolumn (LC-Packings-Dionex, Sunnyvale, CA). Peptide digests were then passed onto a C18 column (75 μm × 150 mm) (LC-Packings-Dionex) and eluted with a gradient from 10% ACN, 0.1% formic acid to 80% ACN, 0.08% formic acid (run duration 60 min, flow rate 200 nl/min). The mass spectrometer was operated in the positive ion electrospray ionization mode with a resolution of 9000 to 11,000 full-width half-maximum. Data-dependent analysis was used for MS/MS (three most abundant ions in each cycle): 1 s MS (m/z 400–1600) and maximum 4 s MS/MS (m/z 50–2000, continuum mode) with 2 min dynamic exclusion. MS/MS raw data were processed using MassLynx 4.0 software (smooth 3/2 Savitzky Golay) (Waters). Peptide identifications from the resulting MS/MS dataset were achieved using an in-house MASCOT server (version 2.0) (Matrix Sciences, London). Full specifications of search parameters are presented in supplemental data files. Quantification was done manually from nano-LC-MS data after integration of peaks for unlabeled/labeled peptide pairs on reconstituted chromatograms obtained by extraction of a specific mass (±0.1 Da) with MassLynx 4.0 software. The minimum signal to noise ratio considered for quantitation was 15:1. SEA and TSST-1 recombinant staphylococcal toxins were submitted to SDS-PAGE and in-gel digestion with trypsin. The peptide digests were analyzed by nano-LC-MS/MS (see Supplemental data files) and nano-LC-MS. Specific tryptic peptides (marker peptides) were selected for each of the two toxins (Table I). Marker peptides were chosen according to their sequence uniqueness among staphylococcal superantigens and their optimal detectability in MS analysis. Three of these marker peptides were made synthesized as AQUA peptides with one [13C6, 15N] l-leucine (mass increase 7 Da).Table IMarker peptides used for the detection and quantification of SEA and TSST-1 staphylococcal superantigenic toxins.Staphylococcal superantigenic toxinSwiss-Prot accession no.Sequence of the specific tryptic peptides used for detection and quantification analysesMonoisotopic mass of the precursorm/z (observed)z (observed)Retention timeminSEAP0A0L2NVTVQELDLQARaSynthesized as AQUA peptide standard.1384.7693.42+27.6QNTVPLETVK1127.6564.82+17.7YNLYNSDVFDGKaSynthesized as AQUA peptide standard.1433.7717.92+32.6TSST-1P06886HQLTQIHGLYR1364.7455.93+15.8LPTPIELPLKaSynthesized as AQUA peptide standard.1119.7560.92+36.3NTDGSISLIIFPSPYYSPAFTK2417.2806.83+29.8QLAISTLDFEIR1404.9703.42+43.0a Synthesized as AQUA peptide standard. Open table in a new tab Commercial SEA and TSST-1 staphylococcal toxins were added in defined amounts into drinking water. Trypsin digestion was performed in-solution. Known amounts of AQUA peptides were added to the peptide digests before nano-LC-MS analysis. For the three unlabeled/labeled peptide pairs selected (Table I), peak doublets separated by a mass of Δm = 7 Da ([13C6, 15N] l-leucine peptides) were observed in the MS survey. Each of these peak doublets was integrated to determine the total ion signal of the natural peptide (mass m) and its corresponding labeled AQUA standard (mass m + Δm). The ratio of these signals allowed a direct calculation of the estimated natural toxin amount which was plotted against the added amount (Fig. 2). Ideally, 100% recovery should have been observed. The SEA and TSST-1 titration curves obtained were linear for added amounts of toxins ranging from 50 to 750 pg. Deviation from linearity above these values resulted from ion signal saturation. The measurements of accuracy and precision were evaluated through the slope values of the titration curves and the standard mean errors (S.E.) values of the data, respectively. AQUA peptide standardization is a highly precise quantification strategy (Fig. 2). However, regarding accuracy, an important discrepancy between the two AQUA peptides targeting SEA was observed (slope value = 1.37 for YNLYNSDVFDGK peptide and slope value = 0.44 for NVTVQELDQAR peptide) (Fig. 2A). Three potentials biases may account for such results: 1) AQUA peptides were quantified by the provider using AAA, obtained lyophilized, and re-solubilized according to the provider's own guidelines. Nevertheless, quantitative re-solubilization should not be taken for granted, and a subsequent evaluation of the re-solubilized peptide by AAA would require much more material than provided. 2) AQUA standards have to be highly diluted prior to their addition into the samples. Depending on their physicochemical properties, dilution of pure peptides can lead to important losses of peptide by adsorption onto vials. 3) Standardization with AQUA peptides does not take into account the yield of the protease digestion step. This step introduces variability due to the intrinsic susceptibility of each protein or protein domain to proteolysis. In addition, variability between samples can also originate from the digestion conditions (e.g. composition of the sample buffer, amount of trypsin, temperature during incubation). Standard peptide adsorption onto vials or incomplete solubilization both lead to an overestimation of the true standard concentration and consequently of the targeted protein. Accordingly, the 37% overestimation of SEA abundance given by the AQUA peptide YNLYNSDVFDGK (slope value = 1.37) likely resulted from incomplete re-solubilization and/or partial adsorption of this standard peptide onto vials. Conversely, the peptides NVTVQELDQAR and LPTPIELPLK may not have been efficiently generated by trypsin digestion and therefore led to an important underestimation of SEA and TSST-1 abundances (slope values = 0.44 and 0.54, respectively). This stresses a major limitation of the AQUA peptide strategy: it does not take into account the actual digestion yield of the different peptides from the native protein. Actually, this drawback is especially problematic for proteins such as staphylococcal superantigenic toxins, which are known to be poorly protease sensitive (15Dinges M.M. Orwin P.M. Schlievert P.M. Exotoxins of Staphylococcus aureus.Clin. Microbiol. Rev. 2000; 13: 16-34Crossref PubMed Scopus (1315) Google Scholar). We designed and produced an isotope-labeled concatemer of staphylococcal superantigenic toxins marker peptides (QconCAT) (Fig. 1A and B). In this cons