ARTICLE pubs.acs.org/jpr
Proteomics under Pressure: Development of Essential Sample Preparation Techniques in Proteomics Using Ultrahigh Hydrostatic Pressure Emily Freeman and Alexander R. Ivanov*,† HSPH Proteomics Resource, Department of Genetics and Complex Diseases, Harvard School of Public Health, Harvard University, 655 Huntington Avenue, SPH-1 Room 409, Boston, Massachusetts 02115, United States
bS Supporting Information ABSTRACT: The minimization of preanalytical variables in sample preparation is imperative for successful discovery-driven and translational research involving large-scale biomolecular profiling. Here, we demonstrate a novel technique using high hydrostatic pressure in addition to several chaotropes and solvents to maximize efficiency of both cell lysis and enzymatic digestion while minimizing the time, manual involvement in sample processing, and preanalytical variability introduced prior to mass spectrometry-based proteomic analysis. The digestion techniques were evaluated and optimized for in-solution, in-gel, and on-membrane applications using protein standards and cell lysates. The lysis techniques were evaluated using human HepG2 cells. Our results demonstrate that the use of elevated pressure and organic solvents can achieve superior protein recovery of organelle-, complex-, and especially membrane-associated proteins, meanwhile obtaining more than a 20-fold increase in throughput with improved reproducibility. This study introduces the concept of ultrahigh-performance sample preparation platforms for targeted characterization of proteome subsets in biological systems. KEYWORDS: in-gel digestion, in-solution digestion, on-membrane digestion, cell lysis, proteomics, pressure cycling technology, fluoroalcohols, sample preparation, mass spectrometry
’ INTRODUCTION Rapid progress in the development of proteomics technologies during the past decade1 3 has been fueled by recent advancements in mass spectrometry (MS) instrumentation, bioinformatics platforms, and the level of expertise, permitting studies demonstrating reproducible interlaboratory characterization of complex biological samples.4 7 Despite these improvements, the current imperfections of discovery-driven proteomics profiling initiatives can be attributed to flawed approaches for sample processing, resulting in the ineffectiveness of sophisticated downstream analytical technologies. In modern proteomics and biological MS techniques, there is a great demand for standardized ultrahigh-performance sample preparation (UHPSP) platforms that can deliver reproducible sample processing in order to recover specific groups of proteins in a targeted manner as well as to enable studies performed by large research consortia. The lysis of a biological sample and the extraction of sample proteins are two of the critical stages of sample preparation in MSbased bottom-up proteomics.8,9 Recent reports have demonstrated the benefits of using pressure and organic solvents for liquid liquid extraction and separation of cells/tissues into distinct fractions of nucleic acids, lipids, subcellular organelles, and proteins.10,11 Techniques such as pressure cycling technology (PCT) are appealing because cell lysis and protein solubilization, r 2011 American Chemical Society
extraction, and denaturation occur synonymously in one thermostatted chamber that is uniformly exposed to alternating cycles of ambient and high hydrostatic pressure up to 240 MPa. It is proposed that this method would avoid variations due to gradient effects and unwanted chemical modifications observed in other extraction methods such as freeze thawing, boiling, sonication, bead beating, and mechanical homogenization. Another crucial sample preparation step for bottom-up proteomics is trypsin-based enzymatic in-solution or in-gel digestion of proteins into peptides of analytical size,12 and the two general protocols have not been radically changed since their introduction approximately 100 and 15 years ago, respectively.13 16 These methods typically require that digestion occurs over a 12 24 h period for optimal results; thus, there is a rate-limiting step in sample preparation. Various modifications have been introduced in order to increase speed and efficiency of proteolysis, including the use of microwave irradiation, ultrasound, or infrared assistance.17 19 Surfactants and other organic solvents have also been added to increase protein accessibility and enhance the proteolytic activity of enzymes.20 26 Hexafluoroisopropanol (HFIP; 1,1,1,3,3,3-hexafluoro-2-propanol) was specifically Received: August 19, 2011 Published: October 26, 2011 5536
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Figure 1. Protocol optimization using the protein mix in PULSE, Eppendorf, and MicroTubes tubes. In plots, white bars correspond to conventional (AP) protocols, and black correspond to PCT. A 100 fmol fraction of the initial protein sample was subsequently analyzed in each replicate LC MS/MS injection. (a) Representation of experiments using protein standards. Three container types were used to test the effect of pressure (PCT), solvents, and reduction components on efficiency and selectivity of enzymatic digestion. (b) Peptide identification results acquired using the protein mix and MicroTubes in various digestion protocols, with and without PCT assistance (n = 6). Fractions of miscleaved standard peptides identified are shown as portions of the total standard protein peptides identified (black). Error bars indicate standard deviations here and in figures below. (c f) Reproducibility for AP and PCT-assisted digests was assessed measuring peptide abundances using the label-free AMT approach. (g) Peptide abundances for PCT digests plotted versus AP digests.
chosen because fluorinated alcohols have been shown to aid in solubilization and extraction of hydrophobic proteins and subcellular protein complexes.10,16,27 Elevated pressure is a variable that has only recently been examined as an accelerating factor in the enzymatic digestion of relatively large concentrations of proteins.23 Though the benefits of high-pressure and an aqueous buffer environment have been briefly illustrated before,21,23,24 a systematic attempt at method optimization for MS-based proteomics applications has not yet been made to our knowledge. We hypothesized that ultrahigh pressure could change protein conformation enough to make the polypeptide residues more accessible to proteolytic cleavage as well as to enhance solubilization of hydrophobic proteins and complexes, synergistic with the stimulating catalytic activities of proteases.24 It was also predicted that PCT- and solvent-assistance may enhance protein and peptide diffusion through the gel matrix, meanwhile minimizing manual intervention and the introduction of additional contaminants at various stages of in-gel protocols. Because of the popularity of protocols that immobilize proteins and peptides to polymeric membranes in immunoaffinity applications compatible with proteomic profiling, it was also of interest to examine onmembrane digestion protocols. We have evaluated the use of PCT in both cell lysis and enzymatic digestion. The protein quantities and concentrations used in our experiments were designed to reflect levels observed in real-world proteomics studies. In addition to pressure, we also examined the effect of several chaotropic agents, organic solvents, and reducing agents. The in-solution digestion techniques were tested and optimized using a mixture of protein standards and then applied to in-gel and on-PVDF (polyvinylidene fluoride) membrane applications using
Schizosaccharomyces pombe cell lysates. The efficiency of digestion was assessed using qualitative and quantitative proteomics profiling techniques. The pressure- and fluoroorganic solvent-assisted lysis techniques were evaluated using human hepatocellular carcinoma (HepG2) cultured cells and compared to conventional methods in their efficiency of protein recovery through characterization of enriched functional categories of MS-detected proteins.
’ EXPERIMENTAL PROCEDURES Optimization of Digestion Protocols for a Mixture of Protein Standards
A “protein mix” of eight standard proteins (Table S1, Supporting Information) was made using an equimolar ratio of 1 pmol/ μL of each protein for protocols in Figure 1 and Table S2 (Supporting Information). Samples to be run with pressure used 1.4 mL PULSE tubes (Pressure BioSciences), and samples for conventional digest conditions used 1.5 mL Eppendorf tubes. Details are in the Supporting Information; in summary: a 5 μL aliquot of the protein mix was mixed with applicable solubilizing agents (HFIP, urea, methanol) at appropriate concentrations (Table S2, Supporting Information) and was then reduced, alkylated, and digested with and without pressure assistance. For proteolysis with trypsin or lysyl endopeptidase (Lys-C), a 1:100 w/w enzyme to substrate ratio was used. Samples were digested at atmospheric pressure (AP) or at 138 MPa. The final E/S weight ratio was 1:50 for trypsin and 1:100 for the Lys-C. Proteolysis was halted with formic acid (0.1%). Protein mix samples were also subjected to variations in total time of digestion (with and without pressure), reduction with and without pressure
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Journal of Proteome Research assistance, as well as choice and concentration of reducing agents. Details are available in the Supporting Information. Optimization of Protocol in MicroTubes
Optimal protocols from the initial trials using PULSE and Eppendorf tubes (see the Supporting Information) were modified for small volume applications using MicroTubes, chemically inert polypropylene tubes of 150 μL volume that can withstand extended temperature and pressure ranges. In triplicate, each sample used 5 μL of protein mix (5 pmol of each protein) and was combined with 50% HFIP, 8 M urea, or 25% methanol, where applicable. Digestion followed nearly identical protocols as those described above (details given in the Supporting Information). Digestion of S. pombe Gels and PVDF Membranes
S. pombe cultures were grown as previously described.28 Cell lysis was conducted in RIPA (radioimmunoprecipitation assay) lysis buffer, and protein concentration was determined (DC assay, BioRad). Approximately 4.0 mg of protein was applied to each of two SDS-PAGE gels; one was immediately electrotransferred to a PVDF membrane (Transblot system, BioRad). After fixing and staining, bands from both the gel and membrane were excised and separated into smaller pieces for analysis of digestion protocols, with and without pressure assistance. All gel samples were processed in PULSE tubes containing an internal 13 mm diameter “lysis disk” that is perforated with 63 holes of 635 μm diameter, whereas membranes were digested in diskless PULSE tubes. Samples were digested as described in method optimization, with the exceptions highlighted in the Supporting Information. Lysis and Digestion of HepG2 Cells
HepG2 cells were grown in MEM (10% FBS) in nine separate 10 cm dishes (plates 1 9). The cells were lysed in 1.0 mL of the appropriate lysis buffer per protocol. Plates 1 4 received the conventional buffer, which contained 8 M urea and protease/ phosphatase inhibitors (detailed information in the Supporting Information). Plates 5 8 were treated with the conventional buffer that also contained 30% HFIP. Plate 9 was treated with 1.0 mL of 100% HFIP as the only lysis reagent. Plates were exposed to appropriate lysis buffers before sonication- or PCTassisted lysis. Six 20 μL aliquots from each of the resulting lysates were transferred to polypropylene MicroTubes and subjected to tryptic digestion with PCT (230 min at 138 MPa) and without PCT (∼36 h at 40 °C). E/S was estimated at a 1:66 w/w ratio. Mass Spectrometry and Data Analysis
Protein identification and MS analyses were performed as described previously29 and in the Supporting Information. In brief, all digests were separated by nanoflow liquid chromatography; the eluent was introduced into the LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific, CA) via nanospray from the tip of the nano-LC column, and the peptide ion species were fragmented using the collision-induced dissociation mode. The database search was performed using the SEQUEST-Sorcerer algorithm (version 4.0.4) and the Sorcerer IDA2 search engine (version 3.5 RC2; Sage-N Research, Thermo Electron, CA). The human FASTA protein sequence database used for searching HepG2 data was processed using the TMHMM Server v. 2.0 (Technical University of Denmark, Lyngby, Denmark) to predict the number of transmembrane domains (TMDs) for each protein. The label-free AMT quantitative analysis was performed using ProGenesis LC MS (version 2.5) software (Nonlinear Dynamics, U.K.), to obtain peptide abundance measurements from the .raw files. For comparative functional and subcellular
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loci characterization of proteins recovered from HepG2 using alternative lysis and digest combinations, we applied the GeneGo MetaCore software suite (Version 6.2, GeneGo Inc., St. Joseph, MI). Other experimental details are provided in the Supporting Information.
’ RESULTS In-Solution Digestion
In our preliminary work, we measured the activity of trypsin at high pressure using a chromogenic substrate, Na-benzoyl-D,Larginine 4-nitroanilide hydrochloride (BAPNA). Sequencing grade N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK)treated modified trypsin turned out to be very stable and did not demonstrate any noticeable losses of activity when exposed to the pressure of up to 139 MPa for several hours.30 To assess the various protocols for digestion of the protein mix (Table S1, Supporting Information), samples were processed using multiple protocols and 1.4 mL PULSE tubes originally designed for PCT applications or 1.5 mL Eppendorf tubes (Figure 1a and Table S2, Supporting Information). To find optimal times for enzymatic digestion, the protein mix was exposed to a protease with or without pressure for varying total times (Figure S1, Supporting Information). In brief, the results showed that PCT-assisted digestion for 45 90 min provided unambiguous identification of all standard proteins in the protein mix, meanwhile decreasing time of digestion at least 20-fold in comparison to conventional overnight, 12, 24, or 40 h digestion. The PCT-assisted samples also showed little difference in enzyme specificity as calculated from miscleaved peptides and reduced the overall percentage of semitryptic peptides when compared to the conventional protocol (Figure S2, Supporting Information). In determining the optimal conditions for reduction of disulfide bonds, on the basis of the number of resulting peptides, we determined that PCT did not provide benefits in the reduction step (Figure S3, Supporting Information) and found that the most effective of the tested reductants was tris(2-carboxyethyl)phosphine (TCEP) (Figure S4, Supporting Information). All further analyses used reduction with 10 mM TCEP and alkylation with iodoacetamide at AP (see the Supporting Information for more details). The experiments performed using PULSE tubes systematically resulted in a general decrease of the total number of identified peptides (Figures S5 S9, Supporting Information). Because of the possibility of the tube design influencing the results of the analyses through sample losses on the tubes themselves or differences in sample handling, an additional set of samples was digested using only MicroTubes and a selection of the best-performing protocols in order to minimize preanalytical variables (see the Supporting Information for more details). As expected, the PCT-assisted digestion in MicroTubes did not result in any decrease in the peptide identification rate while making the digestion throughput up to 20-fold greater. Both PCT-assisted and conventional AP digestion methods provided very similar numbers of identified peptides, and this was observed across all protocols using additional variables, differing only by (1 10% in each case (Figure 1b). Spectral counts and sequence coverage data also showed similar values between versions of the protocols, often slightly higher for PCT-assisted protocols (Figures S9 and S10, Supporting Information). We observed minimal change in the miscleavage rate between AP and the basic PCT protocols (19 38 and 25 29%, respectively; Figures 1b and S11, Supporting Information), and the 5538
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Figure 2. Label-free quantitative LC MS/MS analysis of the resulting digests for the protein mix using the conventional and PCT-assisted digestion protocols. (a) Mean peptide abundances with standard deviations (n = 6) for selected peptide classes. Black and white bars correspond to the conventional and PCT-assisted protocols, respectively. In all figures, an asterisk depicted between two bars denotes significant difference between two sets of samples (p < 0.05, Student’s t-test). Values are mean ( standard error of the mean. (b) The overall peptide abundance ratios. The comparative quantitative analysis was limited to peptides detected in all six LC MS runs. (c f) Examples of peptide abundance measurements using the AMT technique for a peptide identified in several charge states. The peptide DAIPENLPPLTADFAEDK (MW 1954.9523 Da) was identified in two charge states: as 2+ (c and d) and 3+ (e and f) charged ion species. The LC MS1 (single stage MS) peak selection frames used for abundance measurements and the corresponding peak volume values are shown in the panels. The 2+ peptide peak (978.4840 m/z) detected at retention times (RTs) 31.76 ( 0.04 min and 31.85 ( 0.06 min resulted in peak volumes of 8092.3 and 25969.0 for the AP (c) and PCT (d) digestion protocols, respectively. The triply charged peptide peak (652.6586 m/z) detected at RTs 31.75 ( 0.05 min and 31.80 ( 0.06 min resulted in peak volumes of 1986.4 and 7348.1 for the AP (c) and pressure-assisted (d) digestion protocols, respectively. The mean abundance values were used to calculate abundance ratios for the peptides identified within the samples. In the instance of a peptide identified in multiple charge states, the mean value (here 0.29) was taken from the abundance ratios calculated per each of the charge states (0.31 and 0.27, n = 6).
cooperative action of two proteases (Lys-C followed with trypsin) provided the lowest miscleavage rate for both AP and PCT-assisted protocols, which corresponded to 10 15% lower total peptide counts. The highest total peptide counts typically corresponded to protocols providing the highest miscleavage rates (Figures 1b and S11, Supporting Information). Quantitative Assessment of In-Solution Digestion Performance
Evaluations of digestion performance using the numbers of peptide identifications do not provide estimates of the actual changes in peptide abundances. In addition, altered cleavage specificity could lead to additional nontryptic peptides resulting in the increased numbers of peptide identifications. To further assess the performance of digestion, we used the label-free accurate mass and tag (AMT) quantitative approach to analyze the MS data acquired from the protein standards digested in MicroTubes (Figure 2). An example for the AMT quantitation workflow is demonstrated in Figure 2c f. This quantitative analysis showed that overall standard peptide abundance was increased by approximately 25 30% in the PCT digestion protocols (Figure 2a). Further, when the mean peptide peak volume values measured for PCT-derived digests were plotted against conventional method digest values, the correlation coefficient of 0.94 showed strong reproducibility between both data sets (Figure 1g). The average correlation factor (R2) values between individual samples were 0.89 for conventional AP protocols and 0.95 for PCT, whereas the average R2 values for technical digestion replicates assessed using quantitative LC MS runs were 0.88 for AP and 0.93 for PCT, indicating the slightly higher reproducibility in digestion efficiency reflected by peptide abundance for the PCT samples (Figure 1c f, Table S3, Supporting Information). The separation reproducibility
assessed using LC MS replicates was identical for AP and PCT samples and resulted in R2 values of approximately 0.95. The frequency of peptide coelution evaluated at the stage of quantitative analysis was alike for AP and PCT samples; therefore, no obvious differences in ionization efficiency and ion suppression were detected between the protocols. The slope of the trendline comparing mean peptide abundances in AP and PCT digest methods equal to 1.21 also indicated ∼20% higher abundance for PCT-produced digests (Figure 1g). The ratio of conventional/ PCT peptide abundance values indicates that over two-thirds of all identified peptides were found at higher abundance with PCT-assisted digestion in comparison to the AP samples (Figure 2b). We also noted that although PCT showed a higher overall recovery of hydrophobic and hydrophilic peptides alike (measured using the Kyte Doolittle (GRAVY) scores,31 Figure 2a), pressure assistance did not appear to specifically enrich for either type when examining this simple mixture of mostly hydrophilic proteins (see examples in Figure S15b, Supporting Information). Additionally, we were not able to detect any statistically significant changes in abundance of trypsin autolytic peptides with and without pressure. This supported our preliminary data on trypsin’s stability at high pressure. We expected the potential for additional in vitro peptide and protein oxidation induced by high pressure due to the increased pressuredependent solubility of atmospheric oxygen (temperature and all other system parameters affecting oxygen solubility were identical for both protocols); the PCT-assisted samples did generally provide a higher overall number and relative abundance of peptides containing either oxidized M, W, or H residues (Figure 2a), which can be attributed to the overall increased recovery of all peptides in PCT-assisted digestion (Figure S15c, Supporting Information). 5539
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Journal of Proteome Research Despite recent discussions,32 it is generally accepted that trypsin cleaves exclusively at the C-terminal side of R or K residues, except when they are followed immediately by proline (Keil rule).33 We were interested to assess whether changes in digestion protocols affect trypsin specificity; therefore, we tracked the numbers of detected peptides with missed cleavage sites or nonspecific peptide termini (“semi-tryptic”). We found that 17 43% of the total standard peptides identified using both protocol types corresponded to miscleaved peptides when the MicroTubes were used (Figures 1b and S11, Supporting Information). This identification frequency of peptides with missed cleavage sites is very similar to ones previously reported by several prominent groups advocating alternative effectual sample preparation platforms for shotgun proteomics.34 36 Shown with the AMT analysis data, peptides with miscleavages were more abundant in the PCT-assisted digests, demonstrated by the ratio shown in Figures 2a and S15e (Supporting Information). The fully tryptic sibling peptides were the predominant species in the majority of cases (both protocols), and the number of miscleaved variants were often more abundant in PCT when compared to the conventional. This result agrees with our observation made previously regarding the higher abundance of peptides overall using PCT digestion. We observed that ∼90% of all unique peptide identifications possessed fully tryptic termini (FDR e 0.5%), whereas the remaining ∼10% of all unique IDs corresponded to semitryptic peptides and included 75 and 91% of peptide sequences cleaved on N- and C-termini, respectively. Nontryptic peptides were not detected in these experiments (Figures S12 S14, Supporting Information). Among the semitryptic peptides, we observed a higher proportion of “bulky” amino acid residues as the nontryptic termini; 64% of the N-termini corresponded to Y, F, and L residues, and 43% of C-termini were L residues (Figure S13, Supporting Information). This data suggests that tryptic proteolysis may involve some chymotryptic activity (see Supporting Information for more information). It is reasonable to assume that this phenomenon is trypsin batch-dependent or not as pronounced when a mass spectrometer is occupied with fragmenting more numerous sample constituents (“undersampling effect”), as this phenomena has not been observed using samples of higher complexity, both in previous reports34,35,37 and in our subsequent experiments on more complex samples. Using quantitative LC MS data, we have shown that PCT-assisted digestion resulted in a 12% decrease for the total abundance of semitryptic peptides without a change in variability of quantitative measurements compared to values acquired for AP digestion (RSD = ∼7% for both) (Figure S14, Supporting Information). Both examples of semitryptic peptide sequences that are shown in Figure S15b (Supporting Information) are instances of the observed predominant chymotryptic specificity of nontryptic cleavages. Our set of standard proteins contained sequences with a total of eight “KP” and two “RP” sites, and just as the Keil rule dictates,33 we were not able to detect any peptides resulting from the cleavage of the polypeptide chain at these sites. Pressure-Assisted In-Gel Digestion
We examined the use of ultrahigh pressure as an aid for in-gel digestion, specifically to improve enzyme diffusion through the gel, proteolytic hydrolysis, and peptide extraction from the gel matrices. In our experimental design, tubes with a perforated disk were intended to assist in reproducible and automated mincing of the gel pieces into uniform granules approximately 500 600 μm
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Figure 3. Peptide identification results acquired for S. pombe lysates prefractionated on SDS-PAGE and either in-gel digested (a c) or onPVDF-membrane digested after protein transfer by electroblotting (d f). PCT-assisted digestion protocols are compared to conventional. Gel band A corresponds to approximately 35 40 kDa and band B to 45 50 kDa. Unique S. pombe peptides (a) and proteins (b) identified using in-gel digestion. (c) Mean Kyte Doolittle coefficient values for resulting in-gel digests. White and black bars correspond to the conventional and PCT-assisted protocols, respectively.
in diameter and to provide a substantially higher surface-tovolume ratio, as was shown to be beneficial in a previous report.38 On sections of S. pombe loaded gels, we used the same digestion time, pressure, and reduction environment that provided the best results in preliminary experiments. The results show that the use of PCT compared to the conventional AP digestion methods generated similar numbers of peptide IDs for one gel band comparison (band A) (759 ( 40, AP; 728 ( 72, PCT) and a 10% increase in peptide IDs for the second comparison (band B) (756 ( 17, AP; 840 ( 20, PCT) (Figure 3a). The total proteins identified were equivalent in band A for both methods (233 ( 23, AP; 240 ( 16, PCT), and showed a 15% gain for band B when using PCT-assisted digestion (205 ( 9, AP; 237 ( 5, PCT) (Figure 3b). Gel sections used for both protocols were minced using PULSE tubes with perforated disks to minimize sample processing variability. Neither digestion method showed a strong bias toward enhanced recovery of hydrophobic or hydrophilic peptides (Figure 3c). Sample-to-sample reproducibility was not compromised in the higher throughput PCT-assisted methods. The proportion of semitryptic S. pombe peptides identified in AP and PCT did not differ significantly from each other (1.6 ( 0.8 and 1.4 ( 0.3%, respectively, Figure S16, Supporting Information) 5540
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Journal of Proteome Research and did not reveal any evidence of significant chymotryptic activity, with only a slight increase in the mean percentage of miscleaved yeast peptides for PCT compared to conventional (37 ( 9%, PCT; 30 ( 4%, AP; Figure 3a). Additionally, we did not observe any significant difference in oxidation patterns. On-Membrane Pressure-Assisted Digestion
We evaluated several approaches for on-membrane digestion using both PCT and conventional AP methods to compare peptide identification yields. We chose the digestion methods incorporating the organic solvents HFIP and MeOH in addition to testing a conventional protocol utilizing the aqueous ammonium bicarbonate buffer.39 Conventional digestion at AP in an aqueous environment, without the addition of any organic solvents, identified an average of 116 ( 13 unique S. pombe peptides from the two sample bands studied (Figure 3d). There were significantly more peptides identified in the organic solventassisted protocols at AP (176 ( 26, HFIP AP [band C]; 408 ( 12, MeOH AP [band D]) (Figure 3d), resulting in a 1.4- and 3.4fold increase compared to the conventional protocol without organic solvents, respectively. The combination of PCT and HFIP provided identification of almost twice as many peptides as the conventional methods for band C (226 ( 28). Interestingly, when digestion was performed under high pressure in the aqueous buffer, recovery of resulting digests was significantly worse when compared to the conventional protocol (PCT + aqueous buffer, 8 ( 4 IDs), a loss in peptide identifications of nearly 95%. Importantly, the combination of MeOH and PCT produced only 25% of the peptides generated using MeOH at AP (105 ( 13 PCT + MeOH; 408 ( 12, MeOH). The two solvents provided opposite results based on the observed digestion efficiency. Both MeOH (25% v/v throughout protocol) and HFIP (initially 50%, diluted to 6% v/v at digestion) provided better peptide recovery than the aqueous buffer alone (32 and 73% increases, respectively). When compared to conventional aqueous conditions, the assistance of pressure cycling resulted in a gain of nearly 30% for the HFIP-containing buffer and a 60% drop for the MeOH-containing buffer. The concentration of 25% MeOH that improves the digestion efficacy for in-solution (both at atmospheric and PCT, Figure 1b) and on-membrane digestion at AP (Figure 3d,e) could actually impair trypsin activity in the pressure-assisted digestion method, requiring further systematic analyses. Surprisingly, organic solvent-assisted and pressure-assisted digestion typically resulted in a significant drop of the overall average hydrophobicity indexes as compared to the conventional method (Figure 3f); we attribute this to the increases in total peptides recovered using the solventand pressure-assisted methods, where the minute changes in the hydrophobic peptide pool are not obvious amidst the overwhelmingly hydrophilic peptide population (see more details in the Supporting Information). We also do not exclude the possibility of additional losses of the more hydrophilic proteins during electrotransfer. Organic solvent- and PCT-assistance in on-membrane tryptic digestion did not result in losses of enzyme specificity and efficiency, shown by the percentages of total identified peptides that were miscleaved or semitryptic (Figures S17 and S18, Supporting Information). PCT assistance improved the throughput and efficiency of digestion in concordance with the in-solution and in-gel experiments. Pressure-Assisted Cell Lysis
We chose to examine the effect of pressure and organic solvents on protein recovery for the lysis of HepG2 cells, in
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comparison to more traditional osmosis-based sonication lysis techniques (Figure 4a). We also compared the use of three different lysis buffers in order to assess the effect of a fluorinated alcohol (HFIP) on protein recovery: a conventional buffer containing 8 M urea, a 30% HFIP version of the conventional buffer, and 100% HFIP. We hypothesized HFIP would be beneficial in improving the efficiency of cell lysis because of its ability to rapidly denature, solubilize and unfold proteins and protein complexes as well as to deactivate enzymes including kinases, phosphatases, and proteases. Qualitative Characterization of Lysis and Digestion Conditions on Proteomic Profiling Results. The previously evaluated in-solution digestion protocols were applied to complex HepG2 sample lysates generated using the lysis methods described previously. The results from these analyses showed that PCT digestion produced a marked increase in total unique peptides identified from the same amount of starting cells (protein extracts from approximately 5 000 10 000 cells per LC MS/MS injection), meanwhile decreasing total sample processing time to nearly 10% of that required for conventional digestion. Results showed 1100 ( 267 unique human peptides identified per sample run when using PCT digestion; this was a 58% increase from the conventional digestion method (658 ( 110) (Figure 4c). The use of PCT for cell lysis resulted in an increase of approximately 5% over the conventional method for the total peptide identification rate (673 ( 90), while the PCTassisted lysis and digestion combination resulted in an average 27% gain in the power of proteomics profiling (882 ( 126) without losses in reproducibility between sample preparations (Figures 4b,c, S20, and S21, Supporting Information). Similar results were also observed at the protein and sequence coverage levels (Figures 4b and S22, Supporting Information). When an alternative lysis buffer (30% HFIP) was used, peptide identifications increased by approximately 9% at ambient pressure (707 ( 198). The use of 30% HFIP for sonication lysis in combination with PCT digestion did not change the average total number of identified peptides in comparison to the most conventional method (650 ( 191). Interestingly, the use of the 30% HFIP buffer with PCT lysis also produced an increase of 5% in average total identifications when compared to the conventional protocol (681 ( 265), whereas the use of 30% HFIP with PCT-assisted lysis and digestion produced a 15% decrease in identifications (559 ( 308) (Figure 4c). Similar results were also seen at the protein level (Figure 4b). We also examined the use of 100% HFIP as a lysis buffer; sonication lysis combined with digestion performed either at AP or with PCT assistance provided an increase in the average total peptides identified when compared to the conventional method (conventional/HFIP = +15% (751 ( 407), PCT/HFIP = +8% (701 ( 383)) (Figure 4c). Similar differences in identification rate were also observed at the protein level (Figure 4b). We evaluated the possibility of additional PCTinduced in vitro protein oxidation and observed that the total fraction of oxidized peptides were nearly the same with and without PCT-assistance, supporting our previous observations that the hydrostatic pressure does not promote peptide oxidation (Figure S19, Supporting Information). Miscleaved and semitryptic human peptides were examined to assess enzyme efficiency and specificity, and we observed that pressure assistance in digestion did not increase the fractions of miscleaved peptides, which comprised approximately 30 50% of the total peptide identifications (Figures 4c and S20, Supporting Information). The introduction of partial or pure HFIP as 5541
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Figure 4. Analysis of HepG2 cells using alternative lysis and digestion methods. (a) Schematic of the experimental design. A total of nine 10 cm dishes were grown before lysis using various buffers and sonication or PCT. The lysates were then also digested either at AP or using PCT. The resulting identifications from all variations of lysis and digestion protocols were compared for the following features: (b) total unique human peptides and proportions of miscleaved peptides; (c) total human proteins identified per method; (d) identified proteins containing TMDs; (e) proportions of human peptides which were fully- and semitryptic; and (f) the fraction of detected human peptides with semitryptic on the C- and N-termini.
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a lysis buffer led to a new low average (
