Cryoconservation of Peptide Extracts from Trypsin Digestion of

Article pubs.acs.org/jpr

Cryoconservation of Peptide Extracts from Trypsin Digestion of Proteins for Proteomic Analysis in a Hospital Biobank Facility Jesús Mateos,†,§ Alejandra Pintor-Iglesias,†,§ Patricia Fernández-Puente,† Marta García-Camba,‡ Cristina Ruiz-Romero,† Nieves Doménech,‡ and Francisco J. Blanco*,† †

Rheumatology Division, Proteomics Unit-ProteoRed/ISCIII, INIBIC-Hospital Universitario A Coruña, As Xubias 84, 15006 A Coruña, Spain ‡ Biobanco A Coruña, INIBIC-Hospital Universitario A Coruña, As Xubias 84, 15006 A Coruña, Spain S Supporting Information *

ABSTRACT: We tested a semiautomated protocol for the proper storage and conservation in a hospital biobank of tryptic peptide extracts coming from samples with low and high protein complexity for subsequent mass spectrometry analysis. Lowcomplexity samples (serum albumin, serotransferrin. and alpha-S1-casein) were loaded in replicates in SDS-PAGE and subjected to standard in-gel trypsin digestion. For LC− MALDI−TOF/TOF analysis, purified β-galactosidase and human serum samples were in-solution digested following standard procedures and desalted with C18 stage-tips. In both cases, peptides extracts were aliquoted in individually 2D coded tubes, vacuum-dried, barcode-read, and stored in an automated −20 °C freezer in the Biobank facility. Samples were kept dried at −20 °C until the corresponding time-point of analysis, then reconstituted in the proper buffer and analyzed by either MALDI-TOF/TOF (peptide fingerprinting and MS/MS) or LC−MALDI-TOF/TOF following a highly reproducible pattern to ensure the reproducibility of the results. Protein identification was done with either Mascot or Protein Pilot as search engines using constant parameters. Over a period of 1 year we checked six different time points at days 0, 7, 30, 90, 180, and 365. We compared MS and MS/MS protein score, number of identified peptides, and coverage of the identified proteins. In the low complexity samples, the number of peptides detected gradually decreased over time, especially affecting the MS score. However, two of the three proteins − serum albumin and serotransferrin − were identified by both PMF and MS/MS at day 90. By day 180, only MS/MS identification in some replicates was possible. By LC−MS/MS, β-galactosidase and the most abundant serum proteins were identified with good scores at all time points even by day 365, with no detectable peptide loss or decrease in the fragmentation efficiency, although a progressive decrease in peptide intensity indicates that detection of low abundant proteins could not be optimal after very long periods of time. Our results encourage us to use the biobank facility in the future for long-term storage − up to 3 months − of dried peptide extracts. KEYWORDS: biobank, MALDI-TOF/TOF, cryopreservation, proteomics, Human Proteome Project

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INTRODUCTION

of the same sample is required some weeks or even months later by the user. If the original sample has not been properly conserved, then the user of the facility must get enough of the sample for a new analysis, which is time-consuming, and eventually some problems in terms of reproducibility could arise. Furthermore, recently launched initiatives such as the Human Proteome Project (HPP)14−16 will involve large-scale processing, preservation, and handling of proteomic samples, so establishing standardized protocols for the proper preservation of those samples will be of paramount importance. Biobanking offers the solution for proper storage and conservation of samples in individually coded collection tubes, allowing automated pickup of a specific sample without breaking the cold chain of the others. Furthermore, the automated cryopreservation of peptide extracts in individually 2D coded tubes will allow the creation of a centralized database,

Automated cryopreservation and handling of biological samples in robotic biobank facilities has been improved during the last 10 years1−5 with the aim of achieving the proper conservation of tissues, cell lines, biological fluids, and also biomolecules extracts − proteins, nucleic acids, and so on − derived from them to minimize the sample degradation and to ensure the necessary reproducibility in the later biological experiments. Several studies have analyzed the time-course degradation of proteins6,7 or nucleic acids8,9 stored in a biobank facility, but, to date, few have focused on the proper conservation of processed biomolecules, such as tryptic peptides used for protein identification in proteomics experiments,10,11 and, at least to our knowledge, none has tested the long-term conservation in 2D-coded tubes used in automated facilities. Peptide mass fingerprinting and MS/MS-based protein identification12,13 yields spare peptide extracts that, usually, are not conserved in the proteomics facilities due to lack of storage space. However, in most of the cases, a second analysis © 2014 American Chemical Society

Received: October 19, 2013 Published: February 13, 2014 1930

dx.doi.org/10.1021/pr401046u | J. Proteome Res. 2014, 13, 1930−1937

Journal of Proteome Research

Article

Figure 1. Scheme of the proteomics work-flow followed in the study. Purified serum albumin, serotransferrin, and alpha-S1-casein were resolved by SDS-PAGE and in-gel-digested with trypsin. β-galactosidase and human serum were in-solution-digested with trypsin and desalted with homemade stage tips. Peptide extracts were aliquoted, vacuum-dried, and automated cryopreserved at −20 °C until the corresponding time-point of analysis. Photos of Tempo nanoLC and 4800 TOF/TOF systems courtesy of ABSciex.

organized by users or by kind of sample, that is, low peptide complexity versus high peptide complexity samples, with the end result of an improvement of the overall functioning of the facility. Of course, for this to happen, the peptides should be

conserved and recovered properly after, sometimes, an extended (even months) storage time. We have developed a highly reproducible study to test by MALDI-TOF/TOF and LC−MALDI-TOF/TOF the overtime 1931



Article

The samples were analyzed in a 4800 MALDI-TOF/TOF instrument (ABSciex), and 4000 series Explorer v.4.2 software (ABSciex) was used to generate the spectra and peak list. After manual deposition of 1 μL of mass calibrants (4700 Calibration Mixture, ABSciex), plate model and default calibration of the MALDI plate was done with a laser voltage of 3400 kV and 1000 shots/spectrum. Samples were automatically analyzed in MS mode with a laser voltage of 3800 kV and 1500 shots/spectrum. Automated precursor selection was done using an interpretation method (up to 20 precursors/fraction, signalto-noise lower threshold = 50) with a laser voltage of 4800 kV and 2000 shots/spectrum, excluding trypsin autolysis peptides and other background ions). The CID collision energy range used was medium.

cryopreservation of tryptic peptides extracts coming from different origins, representative of the different kind of samples that are usually handled in the proteomic facilities.

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MATERIAL AND METHODS

Preparation of Crude Peptide Extracts from in-Gel Digested Purified Proteins

Purified serum albumin (P02769), serotransferrin (Q29443), and alpha-S1-casein (P02662) (all from bovine origin, purchased from Sigma-Aldrich) were resuspended in Laemli buffer and loaded in homemade 10% acryl amide gels in replicates (amount per lane: serum albumin, 1.44 pmol; serotransferrin, 1.27 pmol; alpha-S1-casein, 5.1 pmol). After the run was complete, the gel was stained with Coomassie blue (Figure 1). After excluding some of the lanes for the analysis, the bands were randomly assigned to the different time points, manually excised from the gel, and subjected to standard manual in-gel digestion as previously described.12 After 16 h of incubation with trypsin at 6 ng/mL at 37 °C, bands were vortexed and sonicated for 15 min each and crude tryptic peptide extracts were then aliquoted in 2D-coded Micronic tubes (tracker tubes, V-bottom propylene 0.75 mL, Cultek, Madrid, Spain), evaporated in a speed vacuum concentrator, 2D-barcode-scanned, and automatically stored in a robotic freezer (STT1000 KIWI, Liconic Instruments, Mauren, Liechtenstein), except for the sample corresponding to Day 0, which was directly analyzed by mass spectrometry (PMF and peptide fragmentation).

LC−MALDI-TOF/TOF of in-Solution Digested β-Galactosidase

For each analysis, the dried sample for the corresponding time point was automatically thawed, then reconstituted in 25 μL of AcN 2% TFA 0.1%, followed by 15 min of vortexing plus 15 min of sonication and transferred to a nanoLC injection vial. LC−MALDI-TOF/TOF analysis was done basically as previously described.20 Three consecutive injections were done in a nanoLC system (Tempo, Eksigent) by reversephase chromatography into a C18 silica-based column (New Objective, Woburn, MA) with an internal diameter of 75 μm and a pore size of 300 Å. The injection volume was 5 μL, which represents ∼80 fmol on column, and peptides were eluted at a constant flow rate (0.35 μL/min) in a 30 min gradient up to 40% AcN. Eluting peptides were automatically mixed with alpha-cyano (4 mg/mL in 70% AcN, 0.1% TFA) at a flow rate of 1.2 μL/min and deposited on a MALDI LC-plate using a SunCollect (SunChrom) spotter. The chromatograms, composed of 120 spots, each one comprising a 15 s deposition, were then analyzed in a 4800 MALDI-TOF/TOF instrument (ABSciex), and 4000 series Explorer v.4.2 software (ABSciex) was used to generate the spectra and peak list. After manual deposition of mass calibrants, plate model and default calibration of the MALDI plate was done with a laser voltage of 3400 kV and 1000 shots/spectrum. Samples were automatically analyzed in MS mode with a laser voltage of 3800 kV and 1500 shots/spectrum. Automated precursor selection was done using a job-wide interpretation method (up to 12 precursors/fraction, signal-tonoise lower threshold = 50) excluding trypsin autolytic peptides and other background ions, with a laser voltage of 4800 kV and 2000 shots/spectrum and medium CID collision energy range.

Preparation of Peptide Extracts from in-Solution Digested Proteins

Alternatively, 1.5 pmol of Escherichia coli β-galactosidase (purchased from Sigma) and 100 μg of chemically depleted human serum17 were in-solution-digested using standard protocols.18 In brief, the dry protein was diluted in 6 M urea, 2 M thiourea, and 50 mM ammonium bicarbonate buffer, then reduced/alkylated with DTT and IA and digested with trypsin (MS gold Promega) at a ratio of 1:50 trypsin/total protein for 16 h at 37 °C. Samples were then desalted using homemade stage-tips,19 and peptides were aliquoted in 2D-barcoded micronic tubes, dried down in a speedvac, 2D-barcode-scanned, and automatically stored in a robotic freezer (KIWI, Liconic Instruments), with the exception of Day 0 samples, which were directly analyzed by LC−MALDI-TOF/TOF Creation of a Sample Database in the Biobank Facility

Dry samples corresponding to the different time points were deposited in a barcoded 96-well rack (Laborack, Cultek), and the 2D barcodes were scanned using a Multivial CANON 4400F scanner (Cultek). Noraybanks software (Noray Bioinformatics, S.L.U.) was used to create the sample database. The 96-well rack was then automatically introduced to a robotic freezer (KIWI, Liconic Instruments). The samples were kept at −20 °C until the corresponding time point of the analysis.

LC−MALDI-TOF/TOF of In-Solution Digested Human Serum

For each analysis, the dried sample for the corresponding time point was automatically thawed, then reconstituted in 25 μL of AcN 2% TFA 0.1% (15 min vortexing plus 15 min sonication) and transferred to an injection vial. Three consecutive injections were done in a nanoLC system (Tempo, Eksigent). The injection volume was again 5 μL, representing ∼2 μg of digested total serum protein in column, and nanoLC was performed essentially as previously described. Peptides were eluted, and the chromatograms were then analyzed in a 4800 MALDI-TOF/TOF system, as described in the previous section for the β-galactosidase.

Peptide Mass Fingerprinting and Peptide Fragmentation of Purified Proteins

For each analysis, the dried sample for the corresponding time point was automatically thawed and reconstituted in 10 μL of TFA 0.1% (15 min of vortexing plus 15 min if sonication), and 2 μL was deposited on a MALDI plate in quadriplicate. One μL of α-CHCA matrix was then added at 4 mg/mL in 50% acetonitrile, 0.1% TFA. 1932



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Database Searching

PMF and peptide fragmentation data from in-gel digested proteins were exported to Mascot Generic Format (MGF) using the “Launch Peaks to Mascot” tool in the 4000 series Explorer v.4.2 software (ABSciex). Peak lists generated were used for free-access online Mascot searching (www. matrixscience.com) using the following parameters: no taxonomy restriction; enzyme used: trypsin, 1 missed cleavage; fixed modifications: carbamidomethyl (C); variable modifications: oxidation (Met); peptide tolerance: 100 ppm; and fragment tolerance: 0.3 Da; peptide charge: +1. Searching was done against the last SwissProt database release. LC−MALDI-TOF/TOF data from in-solution digested proteins were analyzed using Protein Pilot 4.0 software (ABSciex). Protein Pilot search parameters were as follows: Cys-alkylation: iodoacetamide; digestion: trypsin; ID focus: biological modifications; database: last SwissProt release; species filtering: none; search effort: thorough ID and detection protein threshold unused ProtScore (Conf) > 1.3 (95.0%). The scoring model was defined by the Paragon algorithm. In the case of the high complexity samples, false discovery rate (FDR) was estimated using the “PSPEP on” mode.

Figure 2. PMF identification of in-gel digested proteins. Histogram representation of the summary of the Mascot MS Score (left) and the summary of the number of matched peptides for serum albumin and serotransferrin at the six different time points analyzed.

Serum albumin and serotransferrin were no longer detected by PMF after day 90. At day 0, serum albumin and serotransferrin were identified nicely by peptide fragmentation analysis in all replicates, whereas alpha-S1-casein was identified in two replicates with good MS/MS Mascot scores (serum albumin: ΣMS/MS Score (n = 4): 965, Σ number of matched peptides (n = 4): 32; serotransferrin: ΣMS/MS Score (n = 4): 721, Σ number of matched peptides (n = 4): 41; alpha-S1-casein: ΣMS/MS Score (n = 4): 185, Σ number of matched peptides (n = 4): 6). As a result of the decrease in the number of detected peptides, along the time-course MS/MS analysis, we found a progressive decrease in the MS/MS score. Alpha-S1-casein is no longer identified by peptide fragmentation after day 31. Serum albumin is identified in two of the four replicates by day 180, and serotransferrin is identified by day 365 but only in one out of the four replicates (Figure 3). Previously, Kraut et al.10 reported a poor recovery of hydrophobic peptides after −20 °C storage. Therefore, we proposed to determine if hydrophobic peptides are preferentially lost in our study (Figure 5A). Over time, we indeed detected a progressive loss of very hydrophobic peptides. By day 365, only hydrophilic peptides could be detected. In general, identification by PMF and peptide fragmentation between the individual replicates samples has presented a high variability (note poor results for TRFE identification at day 7 and for ALBU at day 31 in Figures 2 and 3), which is inherent to the MALDI-TOF technique.22,23 Our results indicate that these kinds of peptide extracts could be properly conserved only for midterm periods of time under the conditions of our study. The reasons for the progressive decrease in the peptide detection could be diverse, ranging from adsorption of the peptides to the plastic of the tube to modifications due to the prolonged exposure to the typical contaminants found in these samples. Because we were working with nondesalted crude peptide extracts salts, acryl amide, DTT, and IAA are still present in the sample, and perhaps chemical modifications of the peptides drive to a loss of ionization capacity. Also, we have to keep in mind the fact that the analyzed samples are crude extracts and not an exhaustive peptide extraction with AcN and TFA. Assuming that the protein digestion and peptide extraction yield was 100%, which is utopian, the amount of digested protein deposited onto each spot in the MALDI plate was one-fifth of the amount loaded on each PAGE lane. (See

Statistical Analysis

For each replicate analyzed, the following parameters were recorded from the search engines: PMF from in-gel digested proteins: Mascot MS Score and number of matched peptides; peptide fragmentation from in-gel digested proteins: Mascot MS/MS Score and number of matched peptides; LC−MALDITOF/TOF of in-solution digested β-galactosidase: Protein Pilot unused score, percentage of covered sequence, and number of peptides identified with a confidence greater than 95%; LC−MALDI-TOF/TOF of in-solution digested human serum: Protein Pilot unused score, percentage of covered sequence, and number of peptides identified with a confidence greater than 95% for each detected protein, a percentage of total spectra used for the identification. Peptide hydrophobicity for the tryptic peptides identified in PMF and fragmentation from in-gel digested proteins and in LC−MALDI-TOF/TOF of β-galactosidase was calculated using the online SSRC tool (http://hs2.proteome.ca/ SSRCalc/SSRCalc.html), as described in Krokhin et al.21 Individual data for each parameter studied were recorded in Microsoft Excel datasheets and exported to R Commander using R console. R Commander was used for the statistical analysis of the data using nonparametric tests.

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RESULTS AND DISCUSSION

Peptide Fingerprinting and Peptide Fragmentation of in-Gel Digested Proteins

At day 0, serum albumin and serotransferrin were identified nicely by PMF analysis in all of the replicates, with good MS Mascot Scores (Figure 2 and Supplementary Table 1 in the Supporting Information): serum albumin: ΣMS Score (n = 4): 505; Σ number of matched peptides (n = 4): 43; serotransferrin: ΣMS Score (n = 4): 362; Σ number of matched peptides (n = 4): 40. Alpha-S1-casein was not identified in any replicates by PMF, probably due to its low MW. Along the time-course PMF analysis, a progressive decrease in the total number of peptides was detected, driving to a decrease in the MS Mascot Score of the samples (Figure 2). 1933



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Figure 3. Peptide fragmentation identification of in-gel digested proteins. Histogram representation of the summary of the Mascot MS/MS Score (top) and the summary of the number of matched peptides (bottom) for serum albumin, serotransferrin, and alpha-S1-casein at the six different time points analyzed.

the Material and Methods section.) So this represents, in the best case, roughly 288, 254, and 1000 fmol of serum albumin, serotransferrin, and alpha-S1-casein, respectively. A priori, it is plausible to think that we would be able to have better protein identifications if working with desalted peptide extractions. LC−MALDI-TOF/TOF of In-Solution Digested β-Galactosidase

At day 0, β-galactosidase was identified with good MS/MS scores: average Protein Pilot unused MS/MS Score: 59 ± 0.98, average no. of peptides (Conf > 95): 35.33 ± 1.08. Unlike PMF and fragmentation analysis, in this case. there was not a detectable progressive decrease in the total number of peptides detected or in the fragmentation efficiency along the timecourse analysis (Figure 4), even after 1 year of storage at −20 °C. By day 365, β-galactosidase was identified with excellent MS/MS scores: average Protein Pilot unused MS/MS score: 51.77 ± 0.46; average no. of peptides (Conf > 95): 38.33 ± 1.08. To determine if desalting of the peptide extract could improve the recovery of hydrophobic peptides, we calculated

Figure 4. LC−MALDI-TOF/TOF identification of in-solution digested β-galactosidase. Histogram representation of the average Protein Pilot MS/MS Score, average percentage of sequence coverage and average number of matched peptides for the in-solution digested β-galactosidase at the six different time points analyzed.

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the hydrophobicity of the peptides from β-galactosidase identified in each time point of the analysis. As shown in Figure 5B, hydrophobic peptides are identified properly until

identification improves the ionization of the peptides, leading to an improvement in the reproducibility between the replicates (see the standard deviations in Figure 4). LC−MALDI-TOF/TOF of In-Solution Digested Human Serum

At day 0, up to 29 human proteins were identified in the serum samples (average no. of proteins (n = 3): 28.3 ± 0.4; average no. of peptides (Conf > 95) (n = 3): 172.3 ± 1.78; average percentage of covered sequence (n = 3): 40 ± 2.23; average percentage of the total spectra used for the identification (n = 3) 81.3 ± 1.78). Most of the proteins identified at day 0, were identified along the different time points (Figure 6), and after 1 year at −20 °C,

Figure 6. LC−MALDI-TOF/TOF identification of in-solution digested human serum. Histogram representation of the average Protein Pilot MS/MS Score, average percentage of sequence coverage, the average number of matched peptides, and the average percentage of total spectra used for identification for the in-solution digested human serum at the six different time points analyzed.

up to 26 human proteins were identified in the replicates (average no. of proteins (n = 3): 24 ± 1.08; average no. of peptides (Conf > 95) (n = 3): 150 ± 2.16; average percentage of covered sequence (n = 3): 37 ± 0.69; average percentage of the total spectra used for the identification (n = 3) 81 ± 1.08). We have successfully identified the most abundant serum proteins from in-solution trypsin digestion after 1 year of freezing. The number of peptides detected for these proteins and the fragmentation efficiency are satisfactory at all time points studied, indicating that the desalted peptides of a highly complex protein sample are properly preserved for the long term under the conditions of our study. To investigate if the low-abundant fraction of proteins could be detected after prolonged storage time, we have analyzed the total ion chromatograms for every single serum LC run. (See the figures in Supplementary Table 3 in the Supporting Information.) Whereas total intensity is similar until day 90, by day 180 and day 365, the total intensity decreases substantially, indicating that identification of low abundant proteins could not be optimal after very long periods of time (several months to 1 year). As in the case of the in-solution digested β-galactosidase, the LC peptide fractionation drives to an improvement in the ionization capacity of the peptides, with the final result of very high reproducibility between the individual replicates (see standard deviations in Figure 6).

Figure 5. Hydrophobicity analysis of the peptides detected in the identification of low-complexity samples. Box diagrams representing the hydrophobicity index of single tryptic peptides used for identification of (A) crude extracts from in-gel digested proteins (ALBU, TRFE, and CASA) and (B) desalted extracts from in-solution digested BGAL.

the end of the study, indicating that there is not a decrease in their recovery after prolonged storage at −20 °C. In this case, our results indicate that desalted samples coming from insolution trypsin digestion of proteins could be properly preserved under the conditions of our study for long-term periods of time. The desalted peptide extracts are free of salts and contaminants, and perhaps this is the reason why we have obtained better identification when compared with the in-gel digestions, even when the amount of β-galactosidase injected (80fmol) is much less than that of the proteins analyzed by PMF and fragmentation. Also, the peptide fractionation by the LC system coupled off-line to the MALDI-TOF/TOF 1935



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CONCLUSIONS Standardization of protocols for the cryopreservation of proteins processed for proteomics experiments will be of capital importance in the next years for the successful achievement of the objectives defined in the Human Proteome Project. We have demonstrated in this work that tryptic peptide extractions coming from both low and high protein complexity samples could be properly stored and cryopreserved in a semiautomated biobank facility at −20 °C. We have developed a highly reproducible protocol to test the conservation of the peptides, and we have demonstrated that excellent protein identification by mass spectrometry could be obtained from protein samples processed, trypsin-digested, and stored for periods of time up to 3 months in a biobank following similar procedures to that used for biological samples. Our results will definitely help us and, hopefully, other proteomics groups to the standardization of protocols for the proper cryopreservation of processed proteomics samples, especially in large-scale proteomic initiatives like the HPP.

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REFERENCES

(1) De Souza, Y. G.; Greenspan, J. S. Biobanking past, present and future: responsibilities and benefits. AIDS 2013, 27 (3), 303−12. (2) Vaught, J.; Lockhart, N. C. The evolution of biobanking best practices. Clin. Chim. Acta 2012, 413 (19−20), 1569−75. (3) Malm, J.; Végvári, A.; Rezeli, M.; Upton, P.; Danmyr, P.; Nilsson, R.; et al. Large scale biobanking of blood - the importance of high density sample processing procedures. J. Proteomics. 2012, 76 (Spec No.), 116−24. (4) Malm, J.; Fehniger, T. E.; Danmyr, P.; Végvári, A.; Welinder, C.; Lindberg, H.; et al. Developments in biobanking workflow standardization providing sample integrity and stability. J. Proteomics. 2013, 95, 38−45. (5) Marko-Varga, G.; Végvári, Á ; Welinder, C.; Lindberg, H.; Rezeli, M.; Edula, G.; et al. Standardization and utilization of biobank resources in clinical protein science with examples of emerging applications. J. Proteome Res. 2012, 11 (11), 5124−34. (6) Kisand, K.; Kerna, I.; Kumm, J.; Jonsson, H.; Tamm, A. Impact of cryopreservation on serum concentration of matrix metalloproteinases (MMP)-7, TIMP-1, vascular growth factors (VEGF) and VEGF-R2 in Biobank samples. Clin. Chem. Lab. Med. 2011, 49 (2), 229−35. (7) Pramanik, R.; Thompson, H.; Kistler, J. O.; Wade, W. G.; Galloway, J.; Peakman, T.; et al. Effects of the UK Biobank collection protocol on potential biomarkers in saliva. Int. J. Epidemiol. 2012, 41 (6), 1786−97. (8) Duale, N.; Brunborg, G.; Rønningen, K. S.; Briese, T.; Aarem, J.; Aas, K. K.; et al. Human blood RNA stabilization in samples collected and transported for a large biobank. BMC Res. Notes 2012, 5, 510. (9) Palmirotta, R.; De Marchis, M. L.; Ludovici, G.; Leone, B.; Savonarola, A.; Ialongo, C.; et al. Impact of preanalytical handling and timing for peripheral blood mononuclear cells isolation and RNA studies: the experience of the Interinstitutional Multidisciplinary BioBank (BioBIM). Int. J. Biol. Markers 2012, 27 (2), e90−8. (10) Kraut, A.; Marcellin, M.; Adrait, A.; Kuhn, L.; Louwagie, M.; Kieffer-Jaquinod, S.; et al. Peptide storage: are you getting the best return on your investment? Defining optimal storage conditions for proteomics samples. J. Proteome Res. 2009, 8 (7), 3778−85. (11) Bark, S. J.; Hook, V. Differential recovery of peptides from sample tubes and the reproducibility of quantitative proteomic data. J. Proteome Res. 2007, 6 (11), 4511−6. (12) Shevchenko, A.; Tomas, H.; Havlis, J.; Olsen, J. V.; Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 2006, 1 (6), 2856−60. (13) Strader, M. B.; Tabb, D. L.; Hervey, W. J.; Pan, C.; Hurst, G. B. Efficient and specific trypsin digestion of microgram to nanogram quantities of proteins in organic-aqueous solvent systems. Anal. Chem. 2006, 78 (1), 125−34. (14) Paik, Y. K.; Omenn, G. S.; Uhlen, M.; Hanash, S.; Marko-Varga, G.; Aebersold, R.; et al. Standard guidelines for the chromosomecentric human proteome project. J. Proteome Res. 2012, 11 (4), 2005− 13. (15) Segura, V.; Medina-Aunon, J. A.; Guruceaga, E.; Gharbi, S. I.; González-Tejedo, C.; Sánchez del Pino, M. M.; et al. Spanish human proteome project: dissection of chromosome 16. J. Proteome Res. 2013, 12 (1), 112−22. (16) Legrain, P.; Aebersold, R.; Archakov, A.; Bairoch, A.; Bala, K.; Beretta, L.; et al. The human proteome project: Current state and future direction. Mol. Cell. Proteomics 2011, 10, M111.009993. (17) Fernández-Costa, C.; Calamia, V.; Fernández-Puente, P.; Capelo-Martínez, J. L.; Ruiz-Romero, C.; Blanco, F. J. Sequential depletion of human serum for the search of osteoarthritis biomarkers. Proteome Sci. 2012, 10 (1), 55. (18) Fernández-Puente, P.; Mateos, J.; Fernández-Costa, C.; Oreiro, N.; Fernández-López, C.; Ruiz-Romero, C.; et al. Identification of a panel of novel serum osteoarthritis biomarkers. J. Proteome Res. 2011, 10 (11), 5095−101. (19) Rappsilber, J.; Mann, M.; Ishihama, Y. Protocol for micropurification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2007, 2 (8), 1896−906.

ASSOCIATED CONTENT

S Supporting Information *

Mascot MS and Protein Pilot scores and total ion chromatograms for every serum LC run. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions §

J.M. and A.P.-I. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the ISCIII Networked Proteomics Platform (ProteoRed) and its members for support and helpful discussions, Carolina Fernandez-Costa for providing the serum samples, and Antia Solloso for technical help. This study was funded by grants from Fondo Investigacion SanitariaSpain (CP09/00114, PI11/02397, PI12/00329) and Secretaria I+D+I Xunta de Galicia (10CSA916058PR). J.M. (CA11/ 00050) and P.F.-P. (CA09/00458) are supported by Fondo Investigacion Sanitaria-Spain. C.R.-R. is supported by the Miguel Servet program from Fondo Investigacion SanitariaSpain (CP09/00114). This study is integrated into the chromosome 16 Spanish Human Proteome Project. ProteoRed is part of the Biomolecular and Bioinformatics Resources Platform, PRB2-ISCIII, supported by grant (ISCIII, PT13/ 0001 from the Carlos III National Health Institute).

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ABBREVIATONS LC−MALDI-TOF/TOF, liquid chromatography coupled offline to matrix-assisted laser desorption ionization time-of-flight; TFA, trifluoroacetic acid; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; HPP, Human Proteome Project; ppm, parts per million; PMF, peptide mass fingerprinting; DTT, D-L-dithiothreitol; AcN, acetonitrile; α-CHCA, alpha-cyano-4-hydroxycinnamic acid; IAA, iodoacetamide; CID, collision-induced dissociation 1936



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(20) Mateos, J.; Lourido, L.; Fernández-Puente, P.; Calamia, V.; Fernández-López, C.; Oreiro, N.; et al. Differential protein profiling of synovial fluid from rheumatoid arthritis and osteoarthritis patients using LC-MALDI TOF/TOF. J. Proteomics 2012, 75 (10), 2869−78. (21) Krokhin, O. V.; Spicer, V. Peptide retention standards and hydrophobicity indexes in reversed-phase high-performance liquid chromatography of peptides. Anal. Chem. 2009, 81 (22), 9522−30. (22) Albrethsen, J. Reproducibility in protein profiling by MALDITOF mass spectrometry. Clin. Chem. 2007, 53 (5), 852−8. (23) Jiménez C. R., Huang L., Qiu Y., Burlingame A. L. In-gel digestion of proteins for MALDI-MS fingerprint mapping. Curr. Protoc. Protein Sci. 2001; Chapter 16:Unit 16.4.

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Cryoconservation of Peptide Extracts from Trypsin Digestion of

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