Suppression of Peptide Sample Losses in Autosampler Vials - Journal

Technical Note pubs.acs.org/jpr

Suppression of Peptide Sample Losses in Autosampler Vials Karel Stejskal,† David Potěsǐ l,†,‡ and Zbyněk Zdráhal*,†,‡ †

Research Group Proteomics, Central European Institute of Technology and ‡National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic S Supporting Information *

ABSTRACT: Protein or peptide sample losses could accompany all steps of the proteomic analysis workflow. We focused on suppression of sample adsorptive losses during sample storage in autosampler vials. We examined suppression capabilities of six different sample injection solutions and seven types of autosampler vial surfaces using a model sample (tryptic digest of six proteins, 1 fmol per protein). While the vial material did not play an essential role, the choice of appropriate composition of sample injection solution reduced adsorptive losses substantially. The combination of a polypropylene vial and solution of poly(ethylene glycol) (PEG) (0.001%) or a mixture of high concentrated urea and thiourea (6 M and 1 M) as injection solutions (both acidified with formic acid (FA) (0.1%)) provided the best results in terms of number of significantly identified peptides (p < 0.05). These conclusions were confirmed by analyses of a real sample with intermediate complexity (in-gel digest from sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE)). Addition of PEG into the real sample solution proved to prevent higher losses, concerning mainly hydrophobic peptides, during up to 48 h storage in the autosampler in comparison with a formic acid solution and even with a solution of highly concentrated urea and thiourea. Using PEG for several months was not accompanied by any adverse effect to the liquid chromatography system. KEYWORDS: sample losses, peptide adsorption, LC−MS/MS, PEG

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INTRODUCTION Variability and complexity of samples in proteomics require a number of sample preparation, fractionation, or separation steps prior the final mass spectrometric analysis. Each of the steps could represent a source of sample losses introducing qualitative and quantitative changes, while their extent is unequal for particular sample components. These deviate the final analysis results from reality present at the moment of sample collection implying not only reduced description of the studied system but also the possibility of the generation of false conclusions. The last step in sample processing consists of peptide mixture preparation into the sample vial prior to liquid chromatography−tandem mass spectrometry (LC−MS/MS) analysis. Nonspecific adsorption of peptides on solid surfaces, such as autosampler vials, pipet tips, and instrumentation parts, is a known problem. This problem became of high importance with development of highly sensitive mass spectrometric instrumentation capable of detecting peptides in attomole amounts. Mainly in the case of these low abundance components also, quantitative analysis is accompanied with decreased repeatability due to adsorption losses.1 Moreover, it was reported that adsorption occurs in a relatively short time2 (in 15 min), and prediction of peptide adsorption on specific surfaces, based on their biochemical characteristics, is not reliable.3,4 © XXXX American Chemical Society

Several approaches have been applied to eliminate protein/ peptide adsorptive losses like addition of protein-rich (e.g., bovine serum albumin (BSA))3,5 or surfactant (e.g., sodium dodecyl sulfate (SDS))6 solutions. However these approaches are not beneficial in combination with LC−MS/MS due to competition of added proteins with peptides of interest or due to negative interference of convenient surfactants with the LC system, respectively. Recently, applications of LC−MS compatible acid labile surfactants degraded before the LC− MS/MS analysis have been reported.7,8 Urea and thiourea are also commonly used as effective agents increasing the solubility of proteins from protein pellets or directly from intact samples like cell cultures.9,10 Next to these established approaches, employing several other compounds has been reported in relation with protein/ peptide adsorption. Polypropylene glycol coated glass test tubes were used to reduce losses of the carrier protein for juvenile hormone.11 The coating was achieved by filling the glass test tubes with 1% PEG 20 000 solution followed by extensive washing and drying. Recently, PEG 20 000 or Dextran T-70 was used to improve the peptide yield during the filter aided sample preparation method (FASP).12 Low retention/adsorption polypropylene tubes were recommended to minimize Received: February 28, 2013

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dx.doi.org/10.1021/pr400183v | J. Proteome Res. XXXX, XXX, XXX−XXX

Journal of Proteome Research

Technical Note

Table 1. Vial Characteristics

a

material

abbreviation

volume (mL)a

polypropylene polyethylene polymethylpenteneb glass RSA glass silanized glass Kimshield glass

PP PE PMP GL RSA SIL KIM

0.25 0.30 0.30 0.30 0.30 0.30 0.30

company National Scientific Chromacol MicroSolv National Scientific MicroSolv National Scientific National Scientific

catalog number Company

C4013-11 03-PECV 9532S-0XV C4010-630 9502S-02N-RS C4010-S630 C4010-K630

Company Company Company

Nominal volume according to catalog information. bPMP is also referred to TPX as a trademark of Mitsui Chemicals America, Inc.

Table 2. List of Sample Injection Solutions abbreviation

solution composition

note

FA U U/T DMSO AALSI PEG

0.1% (v/v) FA 5 M urea/0.1% (v/v) FA 5 M urea/1 M thiourea/0.1% (v/v) FA 5% (v/v) DMSO/5% (v/v) FA 0.1% AALS I/1% (v/v) TFA 0.001% (w/v) PEG 20 000/0.1% (v/v) FA

mobile phase A precleaning of solution by RP (C18) precleaning of solution by RP (C18)

carbonic anhydrase 2, β-lactoglobulin, and glutamate dehydrogenase was used (for details see the Supporting Information, Table S1). Stock solution containing 200 fmol/μL of each digested protein was prepared by dissolution of lyophilized B6E in 2.5% FA and 10% ACN and stored at −20 °C in 50 μL aliquots. To get the working concentrations of 0.1 fmol/μL, the stock solution was diluted in the first step by 0.1% FA to a concentration of 2.4 fmol/μL. Subsequently, 0.5 μL of this solution was mixed directly in the autosampler vial with 11.5 μL of SIS, and 10 μL of the total 12 μL were directly injected to the LC−MS/MS system. For all experiments with the model sample, a 1 fmol amount of each digested protein in B6E per injection was selected to avoid a possible masking effect of nonspecific adsorption of peptides caused by a high concentration of sample (>100 fmol/μL).2 The total solution volume (12 μL) was kept fixed to ensure a constant area of the vial surface being in contact with the sample solution (surfacevolume ratio). All solutions except the stock solution were prepared immediately before use, and all liquid handling was done as quickly as possible without time delays. Details on the composition of used solutions are summarized in Table 2. The concentration of FA was set to 0.1% aqueous solution equal to our standard use mobile phase A; the concentration of DMSO was adopted from van Midwoud et al.;1 the AALS I surfactant was prepared according to the manufacturer recommendation (highest concentration not influencing the chromatographic separation); and the concentrations of the urea and PEG solutions were optimized prior to the final comparative experiment (see the Supporting Information, Figures S1 and S2). In both cases, the highest possible concentration not affecting the performance of the LC−MS/ MS system was applied. The comparison of the blank analysis of SIS containing 0.001% PEG/0.1% FA with 0.1% FA is in Figure S3 in the Supporting Information. The U/T solution combined urea (U) at the optimal concentration with 1 M thiourea (T). For selection of suitable SIS, sample was injected to the LC− MS/MS immediately after preparation (0 h) and after 24 h of storage in the vials at 4 °C. Three individual PP vials were used for each SIS and time point. The median of the number of

peptide losses,2 especially during long-term storage.13 Organic solvents (e.g., dimethyl sulfoxide or acetonitrile (ACN)) in combination with plastic autosampler vials were also recommended to increase peptide recovery.1,14 On the contrary, use of organic solvents could result in loss of hydrophilic peptides.14 In the present study, we assessed peptide losses during sample storage in autosampler vials. We evaluated the possibility to suppress the adsorption effects by changing the composition of the sample solution injected into the liquid chromatography coupled with tandem mass spectrometry system (LC−MS/MS) and by use of different material of autosampler vials. As a model sample, a tryptic digest of six bovine proteins (B6E) in the amount of 1 fmol per protein was used for comparative experiments. The optimum injection solution was subsequently examined by using Arabidopsis thaliana sample with intermediate complexity.

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10 min incubation (RT)

MATERIALS AND METHODS

Reagents

Formic acid (FA; for mass spectrometry, ∼98%), dimethyl sulfoxide (≥99.5%), urea (ACS reagent, 99.0−100.5%), thiourea (ReagentPlus, ≥99.0%), and polyethylene glycol (BioUltra, 20 000) were bought from Sigma-Aldrich. Progenta Anionic Acid Labile Surfactant I (AALS I) was purchased from Protea Biosciences. Ultrapure water was produced by Milli-Q Advantage A10 (Millipore). All other chemicals (if not otherwise noted) were purchased from Sigma-Aldrich and were of the highest grade available. Materials

Autosampler vials of seven different materials were tested (list including catalog numbers is given in Table 1). All additional sample handling was performed by using 0.5 mL microcentrifuge tubes with ultrasmooth surface minimizing sample retention (TreffLab) and maximum recovery tips (Axygen). Preparation of Model Sample and Composition of Tested Sample Injection Solutions (SISs)

As a model sample, a tryptic digest of six bovine proteins in equimolar amount (B6E; PTD/00001/63; Michrom) containing bovine serum albumin, α-S1-casein, lactoperoxidase, B



Technical Note

significantly identified peptides (p < 0.05; peptide score ≥ 40) of all proteins in the standard mixture was taken for evaluation.

Nitrogen was used as the nebulizing as well as the drying gas. The pressure of nebulizing gas was 8 psi. The temperature and flow rate of drying gas were set to 300 °C and 6 L/min, respectively, and the capillary voltage was 4.0 kV. The mass spectrometer was operated in the positive ion mode in the m/z range of 300−1500 for MS and 100−2500 for MS/MS scans. Two precursor ions per MS spectrum were selected in data dependent manner for further fragmentation. Extraction of the peak lists from the LC−MS/MS raw data was performed using DataAnalysis 4.1 software (Bruker Daltonik). To prevent sample carryover, the injection system of autosampler and the precolumn was washed by solution of 70% ACN, 20% isopropanol, 9.5% ultrapure water, and 0.5% trifluoroacetic acid after each analysis.

Preparation of the Real Sample

Cell suspension cultures of wild-type A. thaliana (ecotype Landsberg erecta) were maintained in 100 mL of MSMO medium (Sigma-Aldrich) according van Leene et al.15 Cells were harvested 2 days after subculturing, frozen in liquid nitrogen, and stored at −80 °C. Plant material was crushed in liquid nitrogen by a mortar and pestle. Powder was homogenized (1:1; w/v) in lysis buffer (0.5% NP-40, 150 mM NaCl, 0.5 mM EDTA, 10 mM Tris-HCl pH = 7.5, 1% (v/ v) protease inhibitor cocktail for plant cells, and 1% (v/v) phosphatase inhibitor cocktail 2 and 3) and incubated for 1 h at 4 °C. After centrifugation at 20 000 × g for 15 min at 4 °C, supernatant with proteins was collected. Protein concentration was determined using the Bio-Rad protein assay (Bio-Rad). The total proteins (50 μg per line) were separated in 8 parallel lines by 15% T SDS-PAGE. The gel was stained with Coomassie blue (Bio-Rad). Identical protein bands were excised manually from an area at ∼45 kDa from each of the 8 lines. After destaining and washing procedures, each band was incubated with 125 ng of trypsin (sequencing grade; Promega) for 2 h (40 °C, 25 mM ammonium bicarbonate). Tryptic peptides were extracted from gels by 2.5% FA in 50% ACN, and extracts of all bands were pooled, dried, and dissolved again in 2.5% FA in 50% ACN for preserving peptides in solution (see the Supporting Information, Scheme S1). Prior to MS analysis, the sample was diluted 10 times and then 0.5 μL of the diluted sample solution was mixed with 11.5 μL of a particular SIS immediately before use. Tryptic digests were prepared in PP vials with FA (as control), PEG (as optimum SIS), and U/ T. Each variant was prepared and analyzed independently in three replicates. Samples were injected at three time points: immediately after dissolution in individual vials (taken as 0 h), after 24 h, and after 48 h storage in vials. For evaluation, the number of peptides significantly identified (p < 0.01; peptide score ≥45) in at least two replicates were compared. There were about 40 protein groups identified by LC−MS/MS analysis.

Database Search Processing

Peak lists generated from B6E or real sample analyses were subjected to database searching using the protein sequence database Swiss-Prot (taxonomy Mammalia) or NCBInr (nonredundant, taxonomy Arabidopsis thaliana), respectively, and inhouse Mascot Server 2.2 (Matrix Science). Oxidation of methionine, one enzyme miscleavage, and correction for one 13 C atom were set for all searches. In the case of B6E, phosphorylation of serine and threonine and carboxymethylation of cysteine were also set as variable modifications. Variable propionamidation of cysteine was set in the case of real sample analyses. Mass tolerances for peptides and MS/MS fragments were 0.5 Da. The significance threshold in the MASCOT searches of B6E or real sample was set to p < 0.05 (Mascot score ≥ 40) or p < 0.01 (Mascot score ≥ 45), respectively. Results based on three repetitions are expressed as median ± standard deviation.

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RESULTS AND DISCUSSION

Sample Injection Solution Composition

Composition of the SIS can dramatically influence results of the LC−MS/MS analysis.1 It has impact mainly on nonspecific peptide adsorption on the autosampler vial surface and/or chromatographic system parts and peptide trapping efficiency on the trap column. Simultaneously, SIS composition should be also fully compatible with the LC−MS/MS system to maintain its sustainability. To evaluate the effect of different SISs on LC−MS/MS analysis results, we selected six solutions partly on the basis of a literature survey and partly as novel candidates in this context. The composition of particular SISs is detailed in Table 2. An aqueous solution of formic acid (FA), a common sampling solution for peptide mixtures, was used as a control.17 Common polypropylene vials with a 0.25 mL inner volume were used for SIS evaluation. Individual analyses were compared according to the number of identified peptides (p < 0.05, score ≥ 40; Figure 1). The results suggest that prolonged storage time (24 h) of sample in the vial did not affect the LC−MS/MS results significantly, as proved by the analysis of variance (p = 0.9). On the other hand, the composition of SIS had a significant effect on the analysis results even if sample was injected immediately (Figure 1). This is most probably caused by different capabilities of individual SISs to suppress fast adsorption in the sample vial taking place immediately prior to sample injection or adsorption within the injection system of the LC.2 The highest number of peptides was identified when the sample was diluted in PEG (0.001%). There were identified 42 ± 6 peptides at 0 h and 44 ± 6 peptides after 24 h storage (Figure 1). The number of peptides

LC−MS/MS Analysis and Data Processing

All MS/MS analyses were performed under identical conditions using an UltiMate 3000 RSLCnano system (Thermo Scientific) online coupled with a HCTultra PTM Discovery System ion trap mass spectrometer equipped with a nanospray (Bruker Daltonik). After injection (10 out of the total of 12 μL present in the sample vial), peptides were concentrated and desalted on a trap column (100 μm i.d. × 30 mm length; filled with 3.5-μm X-Bridge BEH C18 (Waters)) according to a previously described procedure.16 FA (0.1%) was used as the loading solvent (loading speed 4 μL/min for 6 min). Peptides were then eluted using a water/ACN gradient onto the separation column (75 μm i.d. × 150 mm length; 300 nL/min) filled with Acclaim PepMap RSLC C18 (Dionex). The mobile phases consisted of 0.1% aqueous FA (phase A) and 60% ACN, 30% methanol, and 10% 2,2,2-trifluorethanol acidified with FA (0.1%) (phase B). The gradient elution started at 4% of mobile phase B and increased to 20% in the first 20 min. In the next 15 min step, it increased from 20% to 40% and finally the gradient further increased to 95% of mobile phase B in 5 min and remained at this level for the last 5 min. The analytical column outlet was directly connected to the nanoelectrospray ion source of the mass spectrometer. C



Technical Note

glass (GL), Kimshield glass (KIM), silanized glass (SIL), and RSA glass (RSA) (see Table 1). The basic experimental setup was retained as in previous SIS experiments, but in this case only FA (control) and PEG (the best of SISs tested) were compared. The analyses were performed after 24-h storage of sample solution in particular autosampler vials. Generally, all types of tested vials in combination with PEG solution provided significantly better results (35−78% increase in the peptide number) in comparison with the FA solution. The number of identified peptides displayed broader materialrelated variation for samples prepared with FA than with PEG (see Figure 2). PEG-based samples provided similar results for

Figure 1. Number of significantly identified peptides (p < 0.05) identified in the model sample (corresponding to 1 fmol of each digested protein) after mixing with sample injection solution in polypropylene autosampler vials. Particular solutions were injected into the LC−MS/MS system immediately after mixing (0 h) and after 1 day storage (24 h) at 4 °C. Contribution of peptides derived from particular proteins is shown by shades of gray. Analysis of variance showed no significant effect of storage time (p = 0.91) but a significant effect of the SIS type (p < 0.001). The number of identified peptides in the case of PEG utilization was found statistically different when compared to all other SIS (p < 0.001).

identified in the other SISs was decreased by 34−64%, as compared to PEG. As an example, a list of BSA peptides with significant scores identified in PEG and FA immediately after mixing and 1 day after mixing in the vial is presented in Tables S2 and S3 in the Supporting Information. Generally, loss of longer peptides (with retention times over 22 min) in FA was observed in comparison with PEG. The surfactant AALS I did not have any positive effect on losses of peptides (compared to FA) most probably because its capabilities are lost due to acidic cleavage preceding the injection to MS (the surfactant is LC− MS compatible only after cleavage by 1% TFA). All sample protein components were identified only after solutions of U/ T, DMSO, and PEG were used (Figure 1). However, the negative effect on peptide peak width (most prevalently on early eluting peptides) was observed in the case of DMSO (for detail see the Supporting Information, Figure S4). These effects could be caused by the higher elution power of DMSO (in comparison to water).14 For that reason, DMSO was not included in further experiments with the real sample. In conclusion, our results indicate that significant peptide losses occur immediately after placing the peptide sample into the autosampler vial or, possibly, within the LC system during sample injection and loading on the trap column. We propose the use of PEG (0.001%), which effectively decreases sample losses probably due to competition in adsorption with the peptides, keeps model sample stable at least for 96 h (for detail see the Supporting Information, Figure S2), and it has no adverse effects to the chromatographic separation or the LC system (6 months continuous use).

Figure 2. Number of significantly identified peptides (p < 0.05) identified in the model sample (corresponding to 1 fmol of each protein) after mixing with 0.1% formic acid (FA) and 0.001% polyethylene glycol 20 000 (PEG) in different types of autosampler vials. Particular solutions were prepared in triplicates and injected into the LC−MS/MS system after 1 day of storage at 4 °C. Contribution of peptides derived from particular proteins is shown by shades of gray.

PP, PMP, PE, and SIL (the number of identified peptides was about 42). Three types of glass-based vials (GL, KIM, and RSA) showed lower capability to avoid peptide losses than the other types of vials when PEG was used. In the case of FA in combination with PE or RSA vials, a decrease in the number of identified peptides around 50% was observed as compared to PP. This could be due to different construction of each type of vial resulting in a larger inner surface. Despite the observed differences between results for individual sample vial material, statistical significance was confirmed neither for FA (p = 0.07) nor for PEG (p = 0.09). In summary, the type of the vial material did not play as crucial role as compared to SIS composition especially in combination with PEG. Commonly available PP vials were selected for further experiments as one of the best of the tested materials. Real Sample Analysis

Adsorption of proteins/peptides has been examined in several studies but all of them demonstrated this effect by using quite limited set of proteins or peptides.1−3,6,18 To confirm our results derived from analyses of the model sample, we selected a 1-D gel band (Arabidopsis thaliana) with an intermediate protein complexity (see details in the Materials and Methods). The number of peptides identified in samples prepared with FA solution and immediately injected (0 h) was 14% or 20% lower as compared to U/T or PEG, respectively (see Figure 3).

Vial Material

The material of sample vials or more specifically properties of their inner surface which is in contact with the sample could be another important feature in relation with adsorptive losses. Here, we included three plastic-based and four glass-based commercially available autosampler vials: polypropylene (PP), polyethylene (PE), polymethylpentene (PMP), borosilicate D



Technical Note

Figure 3. The real sample was injected three times in a solution of formic acid (FA; black filled bars), urea and thiourea solution (U/T; hatched bars), and polyethylene glycol 20 000 solution (PEG; blank bars) immediately after mixing (0 h), after 1 day (24 h), and 2 days (48 h) storage at 4 °C. Numbers at top of the bars represent sums of peptides significantly identified (p < 0.01) in two of three analyses under particular conditions.

In contrast to the model sample, the time of storage played a more important role in the case of the FA solution. While the number of identified peptides did not change dramatically over the tested time period for samples in U/T and PEG, the number of peptides was reduced by 34% and 44% after 24 and 48 h of storage using FA, respectively. Complete results and overlap of peptides identified for all three time points and all three solutions are shown using Venn diagrams (Supporting Information, Figure S5). PEG or U/T as SIS showed a high number of unique identifications in comparison with FA in all time points, and no prominent loss of a certain group of peptides was observed. To assess the nature of peptides being likely to be preserved in the solution in the presence of particular SISs, we investigated their retention times. In general, peptides are eluted by reversed-phase chromatography according to their increasing hydrophobicity.19 We observed a significant decrease in the number of hydrophobic peptides (peptides with RT > 25 min) in FA after 24 h in comparison with the other two solutions (see Figure 4). This effect was even more pronounced after 48 h. Similarly, the signal decrease of more retained peptides with increasing storage time was evident in base peak chromatograms (BPCs) from analyses of real sample in FA (Supporting Information, Figures S6−S8). More importantly, despite the comparable identification results from real sample analysis in U/T and PEG solution, there was also striking difference found in BPC from U/T and PEG variants after 48 h storage. The difference was more prominent with increasing storage time in the autosampler vial prior to LC−MS/MS analysis.

Figure 4. Histograms of distribution of peptides in real sample during LC−MS/MS analysis according to their retention time (RT). Real sample was diluted in solutions of formic acid (FA), urea and thiourea (U/T), or polyethylene glycol 20 000 and analyzed immediately after mixing (0 h), after 1 day (24 h), and after 2 days storage (48 h) in polypropylene autosampler vials at 4 °C. Samples were analyzed by using a 50 min gradient. Graphs were constructed by STATISTICA version 10 (StatSoft).

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injection solution for efficient reduction of adsorptive losses of peptides in autosampler vials.

CONCLUSIONS Composition of sample injection solution was found to play important role even if samples were immediately analyzed. Compared to other tested additives, PEG represents a simple and cheap solution improving the overall coverage of real sample. No substantial quantitative losses up to 48 h storage under studied conditions and no compromising of the LC− MS/MS system performance was observed. We therefore propose the use of 0.001% PEG as an additive into the sample

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ASSOCIATED CONTENT

S Supporting Information *

Basic information about proteins in the model sample; peptides of BSA identified immediately (0 h) and 1 day (24 h) after preparation in PEG and FA; scheme of real sample preparation procedure; optimizations of urea and PEG concentrations; comparison of PEG and FA blank samples; EIC chromatoE



Technical Note

carrier protein for juvenile hormone from the hemolymph of the tobacco hornworm Manduca sexta Johannson (Lepidoptera: Sphingidae). J. Biol. Chem. 1976, 251 (16), 4979−4985. (12) Wiśniewski, J. R.; Ostasiewicz, P.; Mann, M. High recovery FASP applied to the proteomic analysis of microdissected formalin fixed paraffin embedded cancer tissues retrieves known colon cancer markers. J. Proteome Res. 2011, 10 (7), 3040−3049. (13) Kraut, A.; Marcellin, M.; Adrait, A.; Kuhn, L.; Louwagie, M.; Kieffer-Jaquinod, S.; Lebert, D.; Masselon, C. D.; Dupuis, A.; Bruley, C.; Jaquinod, M.; Garin, J.; Gallagher-Gambarelli, M. Peptide storage: are you getting the best return on your investment? Defining optimal storage conditions for proteomics samples. J. Proteome Res. 2009, 8 (7), 3778−3785. (14) Vatansever, B.; Lahrichi, S. L.; Thiocone, A.; Salluce, N.; Mathieu, M.; Grouzmann, E.; Rochat, B. Comparison between a linear ion trap and a triple quadruple MS in the sensitive detection of large peptides at femtomole amounts on column. J. Sep. Sci. 2010, 33 (16), 2478−2488. (15) van Leene, J.; Stals, H.; Eeckhout, D.; Persiau, G.; van de Slijke, E.; van Isterdael, G.; de Clercq, A.; Bonnet, E.; Laukens, K.; Remmerie, N.; Henderickx, K.; de Vijlder, T.; Abdelkrim, A.; Pharazyn, A.; van Onckelen, H.; Inzé, D.; Witters, E.; de Jaeger, G. A tandem affinity purification-based technology platform to study the cell cycle interactome in Arabidopsis thaliana. Mol. Cell. Proteomics 2007, 6 (7), 1226−1238. (16) Planeta, J.; Karasek, P.; Vejrosta, J. Development of packed capillary columns using carbon dioxide slurries. J. Sep. Sci. 2003, 26 (6−7), 525−530. (17) Lee, D. G.; Houston, N. L.; Stevenson, S. E.; Ladics, G. S.; McClain, S.; Privalle, L.; Thelen, J. J. Mass spectrometry analysis of soybean seed proteins: optimization of gel-free quantitative workflow. Anal. Methods 2010, 2 (10), 1577−1583. (18) Hyenstrand, P.; Metcalf, J. S.; Beattie, K. A.; Codd, G. A. Effects of adsorption to plastics and solvent conditions in the analysis of the cyanobacterial toxin microcystin-LR by high performance liquid chromatography. Water Res. 2001, 35 (14), 3508−3511. (19) Cowan, R.; Whittaker, R. G. Hydrophobicity indices for amino acid residues as determined by high-performance liquid chromatography. Pept. Res. 1990, 3 (2), 75−80.

grams of selected peptides in DMSO and PEG solutions; Venn diagrams of significantly identified peptides in real sample of A. thaliana; comparison of BPCs of real sample analyzed immediately (0 h), 1 day (24 h), and 2 days (48 h) after preparing in FA, U/T, and PEG. 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]. Phone: +420-54949-8258. Fax: +420-54949-2640. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank to Dr. Šedo for helpful discussion during manuscript preparation and text revision. This work was supported by CEITEC (Central European Institute of Technology) (Grant CZ.1.05/1.1.00/02.0068 funded from the European Regional Development Fund) and by Czech Science foundation (Project No. P206-12-G151).

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ABBREVIATIONS SIS, sample injection solution; B6E, six bovine proteins in equimolar amount; AALS I, anionic acid labile surfactant I; BPCs, base peak chromatograms

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REFERENCES

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