mechanistic investigations of hydrostatic pressure effects...

1
Mechanistic Investigations of Hydrostatic Pressure Effects on Tryptic Digestion Alexander Lazarev 1 ; Vera Gross 1 ; Greta Carlson 1 ; Alexander R. Ivanov 2 ;Scott Walmsley 3 ; Alexey Nesvizhskii 3 1 Pressure BioSciences, Inc. 14 Norfolk Ave., South Easton, MA, USA ; 2 Barnett Institute, Northeastern University, Boston, MA, USA; 3 University of Michigan, 1301 Catherine St., Ann Arbor, MI Copyright 2013 Pressure BioSciences, Inc. For more information please visit www.pressurebiosciences.com References Proteolytic digestion is a fundamental bottleneck of proteomic sample preparation. We present the results of a systematic study to deconvolute pressure effects on protease activity from pressure effects on substrate proteins. Model proteins were digested with and without high pressure and analyzed by high resolution MS/MS on the LTQ-Orbitrap XL. The data suggest that pressure effects on digestion are substrate specific, resulting in greater improvements for proteins that are typically resistant to trypsin. Pressure-based digestion appears to be particularly useful for analysis of tough proteins, providing opportunities for time savings and increasing the reproducibility of quantitative analysis. Materials and Methods Introduction 1. Balny C. Biochimica et Biophysica Acta-Proteins and Proteomics 1764 (2006) 632-639. 2. Delgado A., et al., Ann N Y Acad Sci 1189 (2010) 16-23. 3. Winter R., Dzwolak W. Cell Mol Biol (Noisy-le-grand) 50 (2004) 397- 417. 4. McCoy J, Hubbell WL. Proc Natl Acad Sci USA. 2011; 108(4):1331-6. 5. Gross V, Carlson G, Kwan AT, Smejkal G, Freeman E, Ivanov AR, Lazarev A. J Biomol Tech. 2008; 19(3):189-99. 6. Freeman E, Ivanov AR. J Proteome Res. 2011; 10(12):5536-46 7. Getie-Kebtie M, Lazarev A, Eichelberger M, Alterman M. Anal Biochem. 2011; 409(2):202-12. 8. López-Ferrer D, Petritis K, Hixson KK, Heibeck TH, Moore RJ, Belov ME, Camp DG 2nd, Smith RD. J Proteome Res. 2008; 7(8):3276-81. 9. Lee B, Lopez-Ferrer D, Kim BC, Na HB, Park YI, Weitz KK, Warner MG, Hyeon T, Lee SW, Smith RD, Kim J. Proteomics. 2011; 11(2):309- 18 10. Shevchenko A, Tomas H, Havliš J, Olsen JV, Mann M. Nature Protocols. 2006; 1(6):2856-2860 Conclusions The data suggest that pressure effects on digestion efficiency are substrate protein-specific, affecting to the greater extent proteins (e.g. ubiquitin) that are more resistant to conventional tryptic digestion. Pressure digestion does not appear to negatively affect digestion of “easy” protein substrates such as BSA (no significant decrease in peptide recovery or increase in number of semitryptic or miscleaved peptides). Moreover, pressure-enhanced digestion in contrast to conventional method produces greater number of proteotypic peptides that are reproducibly quantified even when short digestion protocols are employed. Thus, pressure can be used to produce more reproducible digests with higher throughput. Specialized data analysis approach using custom spectral libraries in conjunction with NIST MSQC Pipeline and MS1 peptide intensities obtained directly from raw files by NIST ProMS algorithm offered in- depth analysis of peptide intensities relative to their position in a protein sequence. MSACL 2013 Conference, San Diego, CA. Poster 42 Introduction: Pressure Cycling Technology and Pressure Effects on Biological Macromolecules Pressure is a well-understood thermodynamic parameter orthogonal to temperature. Pressure effects on enzyme activity and protein conformation are very complex and present rich opportunities for research. Indeed, high pressure has been shown to weaken hydrophobic interactions between aliphatic amino acid side chains, while electrostatic interactions are known to be enhanced under pressure [1, 2]. Moreover, main pressure effects on biological macromolecules are attributed to pressure perturbation of the interactions of said molecules with the solvent, leading to reversible partial denaturation of proteins, weakening of lipid bilayers and dissociation of multimeric protein complexes [3]. Pressure acts synergistically with chaotropes and detergents leading to protein denaturation. However, pressure-perturbed proteins have been shown to assume conformational forms drastically different from those resulting from thermal or chemical treatment [4]. Pressure Cycling Technology (PCT) uses alternating high hydrostatic pressure to facilitate thermodynamic perturbation of molecular interactions. PCT sample preparation systems offer specific advantages for tissue and cell lysis and improved recovery of hydrophobic molecules, including integral membrane proteins [5, 6]. These systems also have been shown to modulate enzymatic proteolysis and deglycosylation and improve protein sequence coverage in quality control of protein pharmaceuticals. However, most of the published work to date is based on empirical optimization of high pressure extraction and digestion methods [6-9]. Our intent in this on-going study is to systematically investigate high pressure effects on proteases and substrate proteins within the context of proteomics and sample preparation methods for mass spectrometry analysis. Pressure effects on peptide identification Pressure-Enhanced Protein Digestion Workflow Pressure-Enhanced Digestion of Model Proteins In-solution digestion was performed at 50°C either at ambient pressure (control) or with pressure cycling at 20,000psi using the Barocycler NEP 3229 (Pressure BioSciences). An equimolar mixture of model proteins in 8M urea/50mM ammonium bicarbonate was reduced using conventional method [10], then exchanged into 50mM ammonium bicarbonate with 10% n- propanol using 3kDa MWCO Amicon filters, diluted to 0.05 mg/ml (~0.3uM each) and split into aliquots. Trypsin (sequencing grade, Promega) was added at three different enzyme-to-substrate (E:S) ratios (1:10, 1:50, 1:100) and 100ul aliquots of the reaction mixture (in triplicate) were loaded into PCT MicroTubes. PCT conditions: 50 seconds at 20,000 psi, 10 seconds at atmospheric pressure, per cycle. Control samples were incubated in PCT MicroTubes at 50˚ without pressure. Reactions were stopped at 0.5, 1.0, 2.0, 4.0, and 20 hours by the addition of 5ul of 5% formic acid. High Resolution Nano-LC-MS/MS All digests diluted 1:6 with mobile phase were separated by nanoflow liquid chromatography; the eluent was introduced into the LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific) via nanospray from the tip of the nano-LC column, and the peptide ion species were fragmented using the collision-induced dissociation mode. Data Analysis Pressure effects on tryptic digestion were assessed by extensive data analysis that utilized the NIST_MSQC pipeline (v.1.2.0) and a custom PHP/mySQL data analysis pipeline. Spectra were identified using OMSSA and peptide intensities (MS1) were extracted from the raw files. Subsequently the maximum intensities were calculated using ProMS (NIST, v.May 25,2011). Peptide ion intensities were grouped together to form the basis for detection of reproducible peptide abundances thus accounting for various charge states and/or modifications. Data from these results were then grouped by condition/series and the results were analyzed and plotted using the statistical package R (v.2.14). Control Digestion PCT Digestion Nano-LC-MS/MS and Data Analysis Bovine Serum Albumin, Chicken Ovalbumin, Equine Heart Myoglobin, Bovine Cytochrome C, Bovine Ubiquitin + Promega Sequencing Grade Trypsin PCT MicroTubes High performance BSA peptides (C.V.≤20% ) independent of digestion time 1 MKWVTFISLLLLFSSAYSRGVFRRDTHKSEIAHRFKDLGEEHFKGLVLIAFSQYLQQCPFDEHVKLVNELTEFAKTCVADESHAGCEKSLHTLFGDELCKVASLRETYGDMADCCEKQEP 121 ERNECFLSHKDDSPDLPKLKPDPNTLCDEFKADEKKFWGKYLYEIARRHPYFYAPELLYYANKYNGVFQECCQAEDKGACLLPKIETMREKVLASSARQRLRCASIQKFGERALKAWSVA 241 RLSQKFPKAEFVEVTKLVTDLTKVHKECCHGDLLECADDRADLAKYICDNQDTISSKLKECCDKPLLEKSHCIAEVEKDAIPENLPPLTADFAEDKDVCKNYQEAKDAFLGSFLYEYSRR 361 HPEYAVSVLLRLAKEYEATLEECCAKDDPHACYSTVFDKLKHLVDEPQNLIKQNCDQFEKLGEYGFQNALIVRYTRKVPQVSTPTLVEVSRSLGKVGTRCCTKPESERMPCTEDYLSLIL 481 NRLCVLHEKTPVSEKVTKCCTESLVNRRPCFSALTPDETYVPKAFDEKLFTFHADICTLPDTEKQIKKQTALVELLKHKPKATEEQLKTVMENFVAFVDKCCAADDKEACFAVEGPKLVV 601 STQTALA Peptides with low CV: green= in the CONTROL, blue= in the PCT , teal= in both. Peptides quantified with high accuracy are shown with respect to their location on the native protein structure. . PCT, 30.7% Control, 22.8% BSA: repeatable peptide intensities Ubiquitin BSA BSA: Effect of pressure on repeatable unique peptide IDs. Ubiquitin: Effect of pressure on repeatable unique peptide IDs. C_30 AA Position relative intensity 0 60 0 20 40 60 80 100 C_1Hr AA Position relative intensity 0 60 0 20 40 60 80 100 C_2Hr AA Position relative intensity 0 60 0 20 40 60 80 100 C_4Hr AA Position relative intensity 0 60 0 20 40 60 80 100 C_ON AA Position relative intensity 0 60 0 20 40 60 80 100 P_30 AA Position relative intensity 0 60 0 20 40 60 80 100 P_1Hr AA Position relative intensity 0 60 0 20 40 60 80 100 P_2Hr AA Position relative intensity 0 60 0 20 40 60 80 100 P_4Hr AA Position relative intensity 0 60 0 20 40 60 80 100 P_ON AA Position relative intensity 0 60 0 20 40 60 80 100 EQUINE CYTOCHROME C 0 20 40 60 80 100 -40 -20 0 20 40 -40 -20 0 20 40 0 20 40 60 80 100 -40 -20 0 20 40 -40 -20 0 20 40 0 20 40 60 80 100 -40 -20 0 20 40 -40 -20 0 20 40 0 20 40 60 80 100 -40 -20 0 20 40 -40 -20 0 20 40 0 20 40 60 80 100 -40 -20 0 20 40 -40 -20 0 20 40 C_30 AA Position relative intensity 0 100 0 50 100 150 200 C_1Hr AA Position relative intensity 0 100 0 50 100 150 200 C_2Hr AA Position relative intensity 0 100 0 50 100 150 200 C_4Hr AA Position relative intensity 0 100 0 50 100 150 200 C_ON AA Position relative intensity 0 100 0 50 100 150 200 P_30 AA Position relative intensity 0 100 0 50 100 150 200 P_1Hr AA Position relative intensity 0 100 0 50 100 150 200 P_2Hr AA Position relative intensity 0 100 0 50 100 150 200 P_4Hr AA Position relative intensity 0 100 0 50 100 150 200 P_ON AA Position relative intensity 0 100 0 50 100 150 200 TRYPSIN AS A SUBSTRATE 0 50 100 150 200 -40 -20 0 20 40 -40 -20 0 20 40 0 50 100 150 200 -40 -20 0 20 40 -40 -20 0 20 40 0 50 100 150 200 -40 -20 0 20 40 -40 -20 0 20 40 0 50 100 150 200 -40 -20 0 20 40 -40 -20 0 20 40 0 50 100 150 200 -40 -20 0 20 40 -40 -20 0 20 40 C_30 AA Position relative intensity 0 30 0 100 200 300 400 C_1Hr AA Position relative intensity 0 30 0 100 200 300 400 C_2Hr AA Position relative intensity 0 30 0 100 200 300 400 C_4Hr AA Position relative intensity 0 30 0 100 200 300 400 C_ON AA Position relative intensity 0 30 0 100 200 300 400 P_30 AA Position relative intensity 0 30 0 100 200 300 400 P_1Hr AA Position relative intensity 0 30 0 100 200 300 400 P_2Hr AA Position relative intensity 0 30 0 100 200 300 400 P_4Hr AA Position relative intensity 0 30 0 100 200 300 400 P_ON AA Position relative intensity 0 30 0 100 200 300 400 0 100 200 300 400 -10 -5 0 5 10 -10 -5 0 5 10 0 100 200 300 400 -10 -5 0 5 10 -10 -5 0 5 10 0 100 200 300 400 -10 -5 0 5 10 -10 -5 0 5 10 0 100 200 300 400 -10 -5 0 5 10 -10 -5 0 5 10 0 100 200 300 400 -10 -5 0 5 10 -10 -5 0 5 10 CHICKEN OVALBUMIN BOVINE UBIQUITIN C_30 AA Position relative intensity 0 50 0 20 40 60 80 C_1Hr AA Position relative intensity 0 50 0 20 40 60 80 C_2Hr AA Position relative intensity 0 50 0 20 40 60 80 C_4Hr AA Position relative intensity 0 50 0 20 40 60 80 C_ON AA Position relative intensity 0 50 0 20 40 60 80 P_30 AA Position relative intensity 0 50 0 20 40 60 80 P_1Hr AA Position relative intensity 0 50 0 20 40 60 80 P_2Hr AA Position relative intensity 0 50 0 20 40 60 80 P_4Hr AA Position relative intensity 0 50 0 20 40 60 80 P_ON AA Position relative intensity 0 50 0 20 40 60 80 0 20 40 60 80 -30 -10 0 10 20 30 -30 -10 0 10 20 30 0 20 40 60 80 -30 -10 0 10 20 30 -30 -10 0 10 20 30 0 20 40 60 80 -30 -10 0 10 20 30 -30 -10 0 10 20 30 0 20 40 60 80 -30 -10 0 10 20 30 -30 -10 0 10 20 30 0 20 40 60 80 -30 -10 0 10 20 30 -30 -10 0 10 20 30 C_30 AA Position relative intensity 0 40 0 50 100 150 C_1Hr AA Position relative intensity 0 40 0 50 100 150 C_2Hr AA Position relative intensity 0 40 0 50 100 150 C_4Hr AA Position relative intensity 0 40 0 50 100 150 C_ON AA Position relative intensity 0 40 0 50 100 150 P_30 AA Position relative intensity 0 40 0 50 100 150 P_1Hr AA Position relative intensity 0 40 0 50 100 150 P_2Hr AA Position relative intensity 0 40 0 50 100 150 P_4Hr AA Position relative intensity 0 40 0 50 100 150 P_ON AA Position relative intensity 0 40 0 50 100 150 EQUINE MYOGLOBIN 0 50 100 150 -20 -10 0 10 20 -20 -10 0 10 20 0 50 100 150 -20 -10 0 10 20 -20 -10 0 10 20 0 50 100 150 -20 -10 0 10 20 -20 -10 0 10 20 0 50 100 150 -20 -10 0 10 20 -20 -10 0 10 20 0 50 100 150 -20 -10 0 10 20 -20 -10 0 10 20 BOVINE SERUM ALBUMIN C_30 AA Position relative intensity 0 20 0 100 300 500 C_1Hr AA Position relative intensity 0 20 0 100 300 500 C_2Hr AA Position relative intensity 0 20 0 100 300 500 C_4Hr AA Position relative intensity 0 20 0 100 300 500 C_ON AA Position relative intensity 0 20 0 100 300 500 P_30 AA Position relative intensity 0 20 0 100 300 500 P_1Hr AA Position relative intensity 0 20 0 100 300 500 P_2Hr AA Position relative intensity 0 20 0 100 300 500 P_4Hr AA Position relative intensity 0 20 0 100 300 500 P_ON AA Position relative intensity 0 20 0 100 300 500 0 100 200 300 400 500 600 -10 -5 0 5 10 -10 -5 0 5 10 0 100 200 300 400 500 600 -10 -5 0 5 10 -10 -5 0 5 10 0 100 200 300 400 500 600 -10 -5 0 5 10 -10 -5 0 5 10 0 100 200 300 400 500 600 -10 -5 0 5 10 -10 -5 0 5 10 0 100 200 300 400 500 600 -10 -5 0 5 10 -10 -5 0 5 10 0 50 100 150 200 250 300 350 400 450 500 30m 1h 2h 4h ON Identified Spectra C SpC P SpC 0 50 100 150 200 250 300 350 400 30m 1h 2h 4h ON Peptide IDs C Id's P Id's 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1 30m 1h 2h 4h ON Fraction of total IDs Partial (Semi) Total ID's PT_sample C PT_sample P PT_chymo C PT_chymo P 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 30m 1h 2h 4h ON Fraction of total IDs Fully Tryptic Total ID's FT C FT P FT-near D C FT-near D P 0 0.1 0.2 0.3 0.4 0.5 0.6 30m 1h 2h 4h ON Fraction of total IDs Missed K/R Total ID's MC_mid C MC_mid P MC_end C MC_end P Fraction of identified peptides as a function of digestion time, conditions and cleavage type. Digestion under high pressure produces greater total number of peptide IDs for all proteins studied in shorter runs. Moreover, the pressure-enhanced method generates a greater number of fully tryptic peptides and lower number of missed cleavages in shorter digestion experiments (particularly evident at 2 and 4 hour incubation with enzyme) compared to conventional digestion conditions. Relative intensity by amino acid position for all proteins digested at 1:50 enzyme:substrate ratio, pressure treatment vs. control Legend: BLACK – fully tryptic peptides, RED – peptides with 1 or more miscleavage; Panels below: Relative change by AA position:. GRAY trace – Pressure treatment minus Control, BLACK – fully tryptic peptides, RED – peptides with 1 or more miscleavage. Relative distribution of peptide intensities at different E:S ratios Bovine serum albumin peptides identified with CV≤20% (independent of digestion time) at 1:50 E/S. Pressure- enhanced digestion results in significantly greater intensities for most identified peptides in 30 minute digestion method, thus offering greater throughput for targeted proteomics workflows. Greater number of peptides suitable for reliable quantitative analysis (C.V.≤20%) was obtained in pressure-based digestion workflow compared to control (figure below). These peptides covered 30.7% and 22.8% of BSA sequence, respectively.

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Page 1: Mechanistic Investigations of Hydrostatic Pressure Effects ...pressurebiosciences.com/downloads/publications/...Pressure Cycling Technology (PCT) uses alternating high hydrostatic

Mechanistic Investigations of Hydrostatic Pressure Effects

on Tryptic Digestion Alexander Lazarev1; Vera Gross1; Greta Carlson1; Alexander R. Ivanov2;Scott Walmsley3; Alexey Nesvizhskii3

1Pressure BioSciences, Inc. 14 Norfolk Ave., South Easton, MA, USA ; 2Barnett Institute, Northeastern University, Boston, MA, USA; 3University of Michigan, 1301 Catherine St., Ann Arbor, MI

Copyright 2013 Pressure BioSciences, Inc. For more information please visit www.pressurebiosciences.com

References

Proteolytic digestion is a fundamental bottleneck of proteomic sample preparation. We present the results of a systematic study to deconvolute pressure effects on protease activity from pressure effects on substrate proteins. Model proteins were digested with and without high pressure and analyzed by high resolution MS/MS on the LTQ-Orbitrap XL. The data suggest that pressure effects on digestion are substrate specific, resulting in greater improvements for proteins that are typically resistant to trypsin. Pressure-based digestion appears to be particularly useful for analysis of tough proteins, providing opportunities for time savings and increasing the reproducibility of quantitative analysis.

Materials and Methods

Introduction

1. Balny C. Biochimica et Biophysica Acta-Proteins and Proteomics 1764 (2006) 632-639.

2. Delgado A., et al., Ann N Y Acad Sci 1189 (2010) 16-23.

3. Winter R., Dzwolak W. Cell Mol Biol (Noisy-le-grand) 50 (2004) 397-417.

4. McCoy J, Hubbell WL. Proc Natl Acad Sci USA. 2011; 108(4):1331-6.

5. Gross V, Carlson G, Kwan AT, Smejkal G, Freeman E, Ivanov AR, Lazarev A. J Biomol Tech. 2008; 19(3):189-99.

6. Freeman E, Ivanov AR. J Proteome Res. 2011; 10(12):5536-46

7. Getie-Kebtie M, Lazarev A, Eichelberger M, Alterman M. Anal Biochem. 2011; 409(2):202-12.

8. López-Ferrer D, Petritis K, Hixson KK, Heibeck TH, Moore RJ, Belov ME, Camp DG 2nd, Smith RD. J Proteome Res. 2008; 7(8):3276-81.

9. Lee B, Lopez-Ferrer D, Kim BC, Na HB, Park YI, Weitz KK, Warner MG, Hyeon T, Lee SW, Smith RD, Kim J. Proteomics. 2011; 11(2):309-18

10. Shevchenko A, Tomas H, Havliš J, Olsen JV, Mann M. Nature Protocols. 2006; 1(6):2856-2860

Conclusions

The data suggest that pressure effects on digestion efficiency are substrate protein-specific, affecting to the greater extent proteins (e.g. ubiquitin) that are more resistant to conventional tryptic digestion. Pressure digestion does not appear to negatively affect digestion of “easy” protein substrates such as BSA (no significant decrease in peptide recovery or increase in number of semitryptic or miscleaved peptides). Moreover, pressure-enhanced digestion in contrast to conventional method produces greater number of proteotypic peptides that are reproducibly quantified even when short digestion protocols are employed. Thus, pressure can be used to produce more reproducible digests with higher throughput. Specialized data analysis approach using custom spectral libraries in conjunction with NIST MSQC Pipeline and MS1 peptide intensities obtained directly from raw files by NIST ProMS algorithm offered in-depth analysis of peptide intensities relative to their position in a protein sequence.

MSACL 2013 Conference, San Diego, CA. Poster 42

Introduction: Pressure Cycling Technology and

Pressure Effects on Biological Macromolecules

Pressure is a well-understood thermodynamic parameter orthogonal to temperature. Pressure effects on enzyme activity and protein conformation are very complex and present rich opportunities for research. Indeed, high pressure has been shown to weaken hydrophobic interactions between aliphatic amino acid side chains, while electrostatic interactions are known to be enhanced under pressure [1, 2]. Moreover, main pressure effects on biological macromolecules are attributed to pressure perturbation of the interactions of said molecules with the solvent, leading to reversible partial denaturation of proteins, weakening of lipid bilayers and dissociation of multimeric protein complexes [3]. Pressure acts synergistically with chaotropes and detergents leading to protein denaturation. However, pressure-perturbed proteins have been shown to assume conformational forms drastically different from those resulting from thermal or chemical treatment [4]. Pressure Cycling Technology (PCT) uses alternating high hydrostatic pressure to facilitate thermodynamic perturbation of molecular interactions. PCT sample preparation systems offer specific advantages for tissue and cell lysis and improved recovery of hydrophobic molecules, including integral membrane proteins [5, 6]. These systems also have been shown to modulate enzymatic proteolysis and deglycosylation and improve protein sequence coverage in quality control of protein pharmaceuticals. However, most of the published work to date is based on empirical optimization of high pressure extraction and digestion methods [6-9]. Our intent in this on-going study is to systematically investigate high pressure effects on proteases and substrate proteins within the context of proteomics and sample preparation methods for mass spectrometry analysis.

Pressure effects on peptide identification

Pressure-Enhanced Protein Digestion Workflow

Pressure-Enhanced Digestion of Model Proteins In-solution digestion was performed at 50°C either at ambient pressure (control) or with pressure cycling at 20,000psi using the Barocycler NEP 3229 (Pressure BioSciences). An equimolar mixture of model proteins in 8M urea/50mM ammonium bicarbonate was reduced using conventional method [10], then exchanged into 50mM ammonium bicarbonate with 10% n-propanol using 3kDa MWCO Amicon filters, diluted to 0.05 mg/ml (~0.3uM each) and split into aliquots. Trypsin (sequencing grade, Promega) was added at three different enzyme-to-substrate (E:S) ratios (1:10, 1:50, 1:100) and 100ul aliquots of the reaction mixture (in triplicate) were loaded into PCT MicroTubes. PCT conditions: 50 seconds at 20,000 psi, 10 seconds at atmospheric pressure, per cycle. Control samples were incubated in PCT MicroTubes at 50˚ without pressure. Reactions were stopped at 0.5, 1.0, 2.0, 4.0, and 20 hours by the addition of 5ul of 5% formic acid.

High Resolution Nano-LC-MS/MS All digests diluted 1:6 with mobile phase were separated by nanoflow liquid chromatography; the eluent was introduced into the LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific) via nanospray from the tip of the nano-LC column, and the peptide ion species were fragmented using the collision-induced dissociation mode. Data Analysis Pressure effects on tryptic digestion were assessed by extensive data analysis that utilized the NIST_MSQC pipeline (v.1.2.0) and a custom PHP/mySQL data analysis pipeline. Spectra were identified using OMSSA and peptide intensities (MS1) were extracted from the raw files. Subsequently the maximum intensities were calculated using ProMS (NIST, v.May 25,2011). Peptide ion intensities were grouped together to form the basis for detection of reproducible peptide abundances thus accounting for various charge states and/or modifications. Data from these results were then grouped by condition/series and the results were analyzed and plotted using the statistical package R (v.2.14).

Control

Digestion

PCT

Digestion

Nano-LC-MS/MS and Data Analysis

Bovine Serum Albumin, Chicken Ovalbumin, Equine Heart Myoglobin, Bovine Cytochrome C,

Bovine Ubiquitin

+ Promega Sequencing Grade Trypsin

PCT

MicroTubes

High performance BSA peptides (C.V.≤20% )

independent of digestion time

1 MKWVTFISLLLLFSSAYSRGVFRRDTHKSEIAHRFKDLGEEHFKGLVLIAFSQYLQQCPFDEHVKLVNELTEFAKTCVADESHAGCEKSLHTLFGDELCKVASLRETYGDMADCCEKQEP

121 ERNECFLSHKDDSPDLPKLKPDPNTLCDEFKADEKKFWGKYLYEIARRHPYFYAPELLYYANKYNGVFQECCQAEDKGACLLPKIETMREKVLASSARQRLRCASIQKFGERALKAWSVA

241 RLSQKFPKAEFVEVTKLVTDLTKVHKECCHGDLLECADDRADLAKYICDNQDTISSKLKECCDKPLLEKSHCIAEVEKDAIPENLPPLTADFAEDKDVCKNYQEAKDAFLGSFLYEYSRR

361 HPEYAVSVLLRLAKEYEATLEECCAKDDPHACYSTVFDKLKHLVDEPQNLIKQNCDQFEKLGEYGFQNALIVRYTRKVPQVSTPTLVEVSRSLGKVGTRCCTKPESERMPCTEDYLSLIL

481 NRLCVLHEKTPVSEKVTKCCTESLVNRRPCFSALTPDETYVPKAFDEKLFTFHADICTLPDTEKQIKKQTALVELLKHKPKATEEQLKTVMENFVAFVDKCCAADDKEACFAVEGPKLVV

601 STQTALA

Peptides with low CV: green= in the CONTROL, blue= in the PCT, teal= in both. Peptides quantified with high accuracy are shown with respect to their location on the native protein structure.

.

PCT, 30.7% Control, 22.8%

BSA: repeatable peptide intensities

Ubiquitin BSA

*

BSA: Effect of pressure on repeatable unique peptide IDs.

Ubiquitin: Effect of pressure on repeatable unique peptide IDs.

C_30

AA Position

rela

tive in

tensity

060

0 20 40 60 80 100

C_1Hr

AA Position

rela

tive in

tensity

060

0 20 40 60 80 100

C_2Hr

AA Position

rela

tive in

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060

0 20 40 60 80 100

C_4Hr

AA Position

rela

tive in

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060

0 20 40 60 80 100

C_ON

AA Position

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tive in

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P_30

AA Position

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tive in

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P_1Hr

AA Position

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tive in

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060

0 20 40 60 80 100

P_2Hr

AA Position

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tive in

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060

0 20 40 60 80 100

P_4Hr

AA Position

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tive in

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060

0 20 40 60 80 100

P_ON

AA Position

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tive in

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0 20 40 60 80 100

EQUINE CYTOCHROME C

0 20 40 60 80 100

-40

-20

020

40

Index

-40

-20

020

40

0 20 40 60 80 100

-40

-20

020

40

Index

T

-40

-20

020

40

FT

0 20 40 60 80 100

-40

-20

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40

Index

T

-40

-20

020

40

FT

0 20 40 60 80 100

-40

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Index

T

-40

-20

020

40

FT

0 20 40 60 80 100

-40

-20

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Index

T

-40

-20

020

40

FT

C_30

AA Position

rela

tive in

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0100

0 50 100 150 200

C_1Hr

AA Position

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tive in

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0100

0 50 100 150 200

C_2Hr

AA Position

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0100

0 50 100 150 200

C_4Hr

AA Position

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tive in

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0100

0 50 100 150 200

C_ON

AA Position

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0 50 100 150 200

P_30

AA Position

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0100

0 50 100 150 200

P_1Hr

AA Position

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tive in

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0100

0 50 100 150 200

P_2Hr

AA Position

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tive in

tensity

0100

0 50 100 150 200

P_4Hr

AA Position

rela

tive in

tensity

0100

0 50 100 150 200

P_ON

AA Position

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tive in

tensity

0100

0 50 100 150 200

TRYPSIN AS A SUBSTRATE

0 50 100 150 200

-40

-20

020

40

Index

-40

-20

020

40

0 50 100 150 200

-40

-20

020

40

Index

T

-40

-20

020

40

FT

0 50 100 150 200

-40

-20

020

40

Index

T

-40

-20

020

40

FT

0 50 100 150 200

-40

-20

020

40

Index

T

-40

-20

020

40

FT

0 50 100 150 200

-40

-20

020

40

Index

T

-40

-20

020

40

FT

C_30

AA Position

rela

tive in

tensity

030

0 100 200 300 400

C_1Hr

AA Position

rela

tive in

tensity

030

0 100 200 300 400

C_2Hr

AA Position

rela

tive in

tensity

030

0 100 200 300 400

C_4Hr

AA Position

rela

tive in

tensity

030

0 100 200 300 400

C_ON

AA Position

rela

tive in

tensity

030

0 100 200 300 400

P_30

AA Position

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tive in

tensity

030

0 100 200 300 400

P_1Hr

AA Position

rela

tive in

tensity

030

0 100 200 300 400

P_2Hr

AA Position

rela

tive in

tensity

030

0 100 200 300 400

P_4Hr

AA Position

rela

tive in

tensity

030

0 100 200 300 400

P_ON

AA Position

rela

tive in

tensity

030

0 100 200 300 400

0 100 200 300 400

-10

-50

510

Index

-10

-50

510

0 100 200 300 400

-10

-50

510

Index

T

-10

-50

510

FT

0 100 200 300 400

-10

-50

510

Index

T

-10

-50

510

FT

0 100 200 300 400

-10

-50

510

Index

T

-10

-50

510

FT

0 100 200 300 400

-10

-50

510

Index

T

-10

-50

510

FT

CHICKEN OVALBUMIN

BOVINE UBIQUITIN C_30

AA Position

rela

tive in

tensity

050

0 20 40 60 80

C_1Hr

AA Position

rela

tive in

tensity

050

0 20 40 60 80

C_2Hr

AA Position

rela

tive in

tensity

050

0 20 40 60 80

C_4Hr

AA Position

rela

tive in

tensity

050

0 20 40 60 80

C_ON

AA Position

rela

tive in

tensity

050

0 20 40 60 80

P_30

AA Position

rela

tive in

tensity

050

0 20 40 60 80

P_1Hr

AA Position

rela

tive in

tensity

050

0 20 40 60 80

P_2Hr

AA Position

rela

tive in

tensity

050

0 20 40 60 80

P_4Hr

AA Position

rela

tive in

tensity

050

0 20 40 60 80

P_ON

AA Position

rela

tive in

tensity

050

0 20 40 60 80

0 20 40 60 80

-30

-10

010

20

30

Index

-30

-10

010

20

30

0 20 40 60 80

-30

-10

010

20

30

Index

T

-30

-10

010

20

30

FT

0 20 40 60 80

-30

-10

010

20

30

Index

T

-30

-10

010

20

30

FT

0 20 40 60 80

-30

-10

010

20

30

Index

T

-30

-10

010

20

30

FT

0 20 40 60 80

-30

-10

010

20

30

Index

T

-30

-10

010

20

30

FT

C_30

AA Position

rela

tive in

tensity

040

0 50 100 150

C_1Hr

AA Position

rela

tive in

tensity

040

0 50 100 150

C_2Hr

AA Position

rela

tive in

tensity

040

0 50 100 150

C_4Hr

AA Position

rela

tive in

tensity

040

0 50 100 150

C_ON

AA Position

rela

tive in

tensity

040

0 50 100 150

P_30

AA Position

rela

tive in

tensity

040

0 50 100 150

P_1Hr

AA Position

rela

tive in

tensity

040

0 50 100 150

P_2Hr

AA Position

rela

tive in

tensity

040

0 50 100 150

P_4Hr

AA Position

rela

tive in

tensity

040

0 50 100 150

P_ON

AA Position

rela

tive in

tensity

040

0 50 100 150

EQUINE MYOGLOBIN

0 50 100 150

-20

-10

010

20

Index

-20

-10

010

20

0 50 100 150

-20

-10

010

20

Index

T

-20

-10

010

20

FT

0 50 100 150

-20

-10

010

20

Index

T

-20

-10

010

20

FT

0 50 100 150

-20

-10

010

20

Index

T

-20

-10

010

20

FT

0 50 100 150

-20

-10

010

20

Index

T

-20

-10

010

20

FT

BOVINE SERUM ALBUMIN C_30

AA Position

rela

tive in

tensity

020

0 100 300 500

C_1Hr

AA Position

rela

tive in

tensity

020

0 100 300 500

C_2Hr

AA Position

rela

tive in

tensity

020

0 100 300 500

C_4Hr

AA Position

rela

tive in

tensity

020

0 100 300 500

C_ON

AA Position

rela

tive in

tensity

020

0 100 300 500

P_30

AA Position

rela

tive in

tensity

020

0 100 300 500

P_1Hr

AA Position

rela

tive in

tensity

020

0 100 300 500

P_2Hr

AA Position

rela

tive in

tensity

020

0 100 300 500

P_4Hr

AA Position

rela

tive in

tensity

020

0 100 300 500

P_ON

AA Position

rela

tive in

tensity

020

0 100 300 500

0 100 200 300 400 500 600

-10

-50

510

Index

-10

-50

510

0 100 200 300 400 500 600

-10

-50

510

Index

T

-10

-50

510

FT

0 100 200 300 400 500 600

-10

-50

510

Index

T

-10

-50

510

FT

0 100 200 300 400 500 600

-10

-50

510

Index

T

-10

-50

510

FT

0 100 200 300 400 500 600

-10

-50

510

Index

T

-10

-50

510

FT

0

50

100

150

200

250

300

350

400

450

500

30m 1h 2h 4h ON

Identified Spectra

C SpC

P SpC

0

50

100

150

200

250

300

350

400

30m 1h 2h 4h ON

Peptide IDsC Id's

P Id's

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.1

30m 1h 2h 4h ON

Frac

tio

n o

f to

tal I

Ds

Partial (Semi) Total ID's

PT_sample C

PT_sample P

PT_chymo C

PT_chymo P

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

30m 1h 2h 4h ON

Frac

tio

n o

f to

tal I

Ds

Fully Tryptic Total ID's

FT C

FT P

FT-near D C

FT-near D P

0

0.1

0.2

0.3

0.4

0.5

0.6

30m 1h 2h 4h ON

Frac

tio

n o

f to

tal I

Ds

Missed K/R Total ID's

MC_mid C

MC_mid P

MC_end C

MC_end P

Fraction of identified peptides as a function of digestion time, conditions and cleavage type.

Digestion under high pressure produces greater total number of peptide IDs for all proteins studied in shorter runs. Moreover, the pressure-enhanced method generates a greater number of fully tryptic peptides and lower number of missed cleavages in shorter digestion experiments (particularly evident at 2 and 4 hour incubation with enzyme) compared to conventional digestion conditions.

Relative intensity by amino acid position for all proteins digested at 1:50 enzyme:substrate ratio, pressure treatment vs. control Legend: BLACK – fully tryptic peptides, RED – peptides with 1 or more miscleavage; Panels below: Relative change by AA position:. GRAY trace – Pressure treatment minus Control,

BLACK – fully tryptic peptides, RED – peptides with 1 or more miscleavage.

Relative distribution of peptide intensities at different E:S ratios

Bovine serum albumin peptides identified with CV≤20% (independent of digestion time) at 1:50 E/S. Pressure-enhanced digestion results in significantly greater intensities for most identified peptides in 30 minute digestion method, thus offering greater throughput for targeted proteomics workflows. Greater number of peptides suitable for reliable quantitative analysis (C.V.≤20%) was obtained in pressure-based digestion workflow compared to control (figure below). These peptides covered 30.7% and 22.8% of BSA sequence, respectively.