mechanistic investigations of hydrostatic pressure effects...
Embed Size (px)
TRANSCRIPT
-
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
tensity
060
0 20 40 60 80 100
C_4Hr
AA Position
rela
tive in
tensity
060
0 20 40 60 80 100
C_ON
AA Position
rela
tive in
tensity
060
0 20 40 60 80 100
P_30
AA Position
rela
tive in
tensity
060
0 20 40 60 80 100
P_1Hr
AA Position
rela
tive in
tensity
060
0 20 40 60 80 100
P_2Hr
AA Position
rela
tive in
tensity
060
0 20 40 60 80 100
P_4Hr
AA Position
rela
tive in
tensity
060
0 20 40 60 80 100
P_ON
AA Position
rela
tive in
tensity
060
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
020
40
Index
T
-40
-20
020
40
FT
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
020
40
Index
T
-40
-20
020
40
FT
C_30
AA Position
rela
tive in
tensity
0100
0 50 100 150 200
C_1Hr
AA Position
rela
tive in
tensity
0100
0 50 100 150 200
C_2Hr
AA Position
rela
tive in
tensity
0100
0 50 100 150 200
C_4Hr
AA Position
rela
tive in
tensity
0100
0 50 100 150 200
C_ON
AA Position
rela
tive in
tensity
0100
0 50 100 150 200
P_30
AA Position
rela
tive in
tensity
0100
0 50 100 150 200
P_1Hr
AA Position
rela
tive in
tensity
0100
0 50 100 150 200
P_2Hr
AA Position
rela
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
rela
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
rela
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.