tools for improved protein mass spec sample preparation by promega
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1
Tools for Improved
Protein Mass Spec Sample Preparation
Mourad FERHAT, Ph.D.
15/03/2017
Promega Francemourad.ferhat@promega.com
Promega Corporation
Manufacturer of reagents, kits and integrated systems for life science market
Promega Headquarters Madison, WI
Founded in 1978
1,300 employees in 15 countries
Over 3,500 products
ISO 13485 certified
~ 750 patents
Operations in:
San Luis Obispo, CA
Sunnyvale, CA
Seoul and Shanghai
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Protein fractionation
Mass spec analysis
Protein mass spec sample preparationCommon shortcomings
peptides
Incomplete digestion
Trypsin is not suitable for analysis
Poor peptide recoveryLong and laborious
sample prep procedure
Poor protein extraction fromtissues
Protein extraction
Inadequate instrument performancemonitoring
Protein digestion
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Enhanced Proteolysis with
Trypsin/Lys-C Mix
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Minimized missed cleavages
Increased tolerance to trypsin inhibiting agents
Trypsin, Sequencing Grade
The highest digestion efficiency Tolerance to protease inhibiting agents
Digestion efficiency
Higher digestion efficiency Higher purity (TPCK treatment + affinity
purification) Resistant to autolysis (Lysine residues are
modified by reductive methylation)
Overall good performance
Enhancing of trypsin performance
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Trypsin Gold, Mass Spec Grade
Trypsin/Lys-C mix
What is the nature of incomplete proteolysis in trypsin digests?
Overnight trypsin digest of yeast protein extract
22.2%missedcleavages
Missed R 3.6%
Missed K 18.6%
2.6
4
Majority of missedcleavages occurs atlysine sites.
Missed cleavages
Trypsin cleavage sites
NNNNR NNNNKNNNN
K
RK
K
K K
KK
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R : Arginine K : Lysine
Solution: supplementing trypsin with Lys-C
Lys-C
NNNNK NNNN
Lysines are cleaved with high efficiency.
Lys-C is an ideal means to compensate for trypsin lysine cleavage inefficiency.
Trypsin
NNNN(R/K) NNNN
Lysines are cleaved less efficiently.
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Enhanced proteolysis with Trypsin/Lys-C
Missed R 3.6%
Missed K 18.6%
Trypsin/Lys-C digest
3.6%4%
Overnight digestion at 37oC
Trypsin digest
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Enhanced proteolysis with Trypsin/Lys-C
Missed R 3.6%
Missed K 18.6%
Trypsin digest Trypsin/Lys-C digest
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3.6%4%
Trypsin/Lys-C eliminates majority of missed cleavage sites.
Overnight digestion at 37oC
Study #1: Analysis of FFPE skin tissue
21.5%
8.5%
Trypsin/Lys-C
Missed Cleavages, %
TrypsinTrypsin
Identified Peptides
Trypsin/Lys-C
Identified Proteins
TrypsinTrypsin/Lys-C
24% increase 10% increase2.5 fold drop
705887
165
10
182
Sample prep is difficult due to extensive protein crosslinking in FFPE tissue.
Courtesy of Chris Adams, Stanford U
Trypsin/Lys-C increased number of identified peptides and proteins in FFPE tissue.
Study #2: Developing biomarker quantitative assay for human plasma
Courtesy of Matt Szapacs, GSK
674099
Trypsin/Lys-C digest
9139
Trypsin/Lys-Cdigest
Trypsin digest
3743
Trypsin/Lys-C digest
1180
Trypsin/Lys-Cdigest
TrypsindigestTrypsin
digest Peptide peak area
Trypsin digest
SAP protein
145305
8976
2207 555
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Trypsin/Lys-C provided conditions for more accurate quantitation of the targeted protein in plasma.
Study #3: Increased tolerance to trypsin inhibiting agents
Inhibitor: GuClInhibitor: proteaseinhibitor cocktail
1252
1495
Trypsin/Lys-C mix assures efficient proteolysis even if a proteinsample is contaminated with trypsin inhibiting agents.
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Inhibitor Protease Missed cleavages
Protease inhibitorcocktail, 1X
Trypsin 44.4%
Trypsin/Lys-C 21.5%
GuCl, 0.5 MTrypsin 55.9%
Trypsin/Lys-C 24.6%
Digestion of yeast protein extract containing trypsin inhibiting agents
Missed (undigested) cleavage sites Identified proteins
Trypsin
Trypsin/Lys-C
Trypsin
13-20% increase
1364
1204
Trypsin/Lys-C
Advantages of Alternative Proteases
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Alternative cleavage specificity
Activity under trypsin-inhibiting conditions
Alternative cleavage specificity and reactionconditions
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Protease Cleavage site Property and application
Lys-C NNNNK NNN Active under denaturing conditions Digest proteolytically resistant proteins
Glu-C NNNNE NNN Used as trypsin alternative if trypsin cleavagesites have disadvantageous distribution
Asp-N NNNN DNNN
Arg-C NNNNR NNN(also cleave at K at a lesser degree)
Analysis of histone posttranslationalmodifications
Chymotrypsin NNNN(F/Y/W) NNN Digests hydrophobic proteins (i.e. membrane proteins)
Pepsin Nonspecific protease Works at low pH Used in HDX studies
Thermolysin Nonspecific protease Works at elevated temperature Digest proteolytically difficult proteins;
structural studies
Elastase Nonspecific protease Used to increase protein coverage
Pepsin and thermolysin are a better alternative for membrane proteins than trypsin
Fully digest membrane proteins
Low pH and high temperature used by these proteases help unfolding these proteins.
Case study: digestion of membrane proteins
Too few tryptic cleavage sites
Tight folding prevents trypsinaccess to cleavage sites
15
20
0
Bacteriorhodopsin coverage was dramatically increased whendigested with thermolysin and pepsin.
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40
60
100
80
Pepsin
Thermolysin
Trypsin
Seq
ue
nce
cove
rage
,%
Bacteriorhodopsin
Coverage with trypsin
Coverage with pepsin
Bacteriorhodopsin sequence coverage
Increased protein sequence coverage with pepsinand thermolysin
IdeS – Immunoglobulin Degrading Enzyme fromStreptococcus pyogenes
IdeS is an IgG-specific protease. It cleaves IgG at a unique site below the hinge.
’
Fc/2 (+Glycans)
LC
Mass spec
30 min digestion
Fd’
IdeS
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IdeS advantage for IgG characterization
Ready separation of IgG Fragments Rapid analysis of major protein modifications
IdeZ and IdeS protease cleavage specificity
We have recently added IdeZ protease to ourmass spec reagent portfolio. IdeZ offers furtherimprovement for IgG analysis.In contrast to IdeS, which preferentially cleaveshuman antibodies, IdeZ also efficiently cleavesmouse antibodies.
Characterization of Protein Glycosylation
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PNGaseF
EndoH
Analysis of Glycoproteins with LC/MS and PNGase F
Asn-linked type glycans can be cleaved enzymatically by PNGase F :
The cleavage separate intact oligosaccharides from slightly modified proteins(Asn residues at the site of N-glycosylation are converted to Asp)
The deglycosylated peptides can be analyzed by mass spectrometry
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Using EndoH and PNGase F to monitor protein
trafficking
Protein Digestion in Gel with MS Compatible
Surfactant
Promega Corporation 19
Increased peptide recovery
In-gel digestion and peptide extraction in a single 1h step
In-gel protein digestionAdvantages and challenges
Advantages of SDS-PAGE protein fractionation
Rapid removal of mass spec interfering impurities
Efficient reduction of sample complexity
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Shortcomings of in-gel protein digestion
Inefficient peptide recovery from gel
Extensive peptide loss due to adsorption to a plastic ware
Lengthy and laborious procedure
In-gel protein digestionAdvantages and challenges
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ProteaseMAX™ Surfactant
Cleavable bonds
Degradation by temperature or acid
LC/MS compatible compounds
Zwitterionic headHydrophobic tail+
Degradation Products
ProteaseMAX™ mass spec compatible surfactant
Mass spec compatible anionic surfactant
Cleavable bonds
Self-degradable mass spec compatible surfactant
ProteaseMAX™ is designed to self-degrade over the course of mass specprotein sample preparation onto mass spec innocuous compounds.
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Peptide Increase in peptide recovery withProteaseMAX™, fold
AGGALCANGAVR 1.45QGDDGAALEVIEVHR 2.06
EHLPLPSEAGPTPCAPASFER 1.80
Improved peptide recovery
ProteaseMAX™ increases peptide recovery from gel.
MALDI-TOF spectrum of HTR1A protein digested in gel
Peptides recovered with ProteaseMAX™
Peptides recovered in conventional digestion
Saveliev et al. Analytical Chemistry 2013, 85 (2), pp 907–914.
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Minimized peptide adsorption to plastic ware
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Peptide Increase in soluble peptide with ProteaseMAX (fold)
PLSRTLSVAAK 16.6
TTYADFIASGRTGRRNAIHD 9.2
AAKIQASFRGHMARKK 4.6
EPPLSQEAFADLWKK 2.05
Saveliev et al. Analytical Chemistry 2013, 85 (2), pp 907–914.
ProteaseMAX™ minimizes peptide adsorption to a plastic ware.
Enhanced analysis of a complex protein mixture withProteaseMAX™-assisted in-gel digestion
Gel-LC Analysis of Mouse Protein Extract
Courtesy of Dr. Chris Adams, Stanford U
ProteaseMAX™ increases number of peptide and proteinidentifications in a cell extract digested in gel.
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Conventional In-gel Protein Digestion
Peptide extraction(1.5 – 2 h)
Mass spec analysis Mass spec analysis
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In-gel Protein Digestion withProteaseMAX™
Digestion/extraction step(1 h)
Protein digestion and peptide extraction are complete in a single 1 h step.
12-18 h
Streamlined and rapid in-gel digestion withProteaseMAX™
Protein Extraction from Tissues with MS
Compatible Surfactant
Promega Corporation 27
MS compatible SDS analog for tissue proteomics
Efficient extraction of hydrophobic proteins
Surfactant 3273 – MS compatible SDS analog fortissue proteomics
Surfactant 3273
Degradation by a strong acid
Cleavable bonds
Mass spec compatible anionic surfactant
Degradation Products
Hydrophobic tail Zwitterionic head
+
LC/MS compatible compounds
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3273 is designed for efficient protein extraction from tissues and other biological samples and solubilization of protein pellets.
Enhanced protein extracting and solubilizing capability Tolerates harsh treatment, including boiling
Improved protein extraction from pig heart withsurfactant 3273
Protein IDs in pig liver extractsTotal extracted protein
SDS-PAGE
Chang et al. J. Proteome Res. 2015, 14 (3), pp 1587–1599.
RapiGest
Control
3273
Control
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Total membrane protein IDs in tissue extracts
3273
3273 enhances protein extraction from animal tissues. Protein extraction efficiency is comparable to SDS. Number of recovered membrane proteins is significantly increased.
Reference MS Protein Materials
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Validated test material for sample prep optimization
Standards for monitoring all key LC and MS performance parameters
Highly complex reference protein material for:
Mass spec instrument performance monitoring
Sample preparation method development
Features
Compatible with LC/MS
Pre-processed for immediate use
Lot to lot consistency in protein composition and abundance
Provided in intact and pre-digested formats.
MS-compatible whole cell protein extractsModel proteomic material
K562 human cells
Yeast
Reference Protein Materials address the critical question:Do my mass spec instrument, reagents and method work properly?
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Mass spec instrument performance monitoring
RT: 0.00 - 106.14 SM: 5G
0 30 40 50 60 70 80 90 100
Time (min)
40
20
0
80
60
100
0
40
20
80
60
100
40
20
0
80
60
10087.6345.71
38.7423.45 82.0432.20 35.72 79.9543.26 52.11 58.3726.04 61.02 77.63 90.0665.57 98.66
21.09
15.17 19.385.10
44.31
87.3730.83
37.51
41.9722.59 81.30 81.9649.48 50.78 57.2021.37 89.7775.56 76.69
97.6218.5711.088.74
38.22
45.1087.9122.92
31.60 82.6635.2025.48 58.4548.88 82.4565.43
72.4396.42 98.61
20.61
14.88 18.94
10 20
8.10
st
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1 week
nd2 week
3rd week
Detecting deterioration of an instrument performance in a timelyManner (1 µg of human predigested protein extract)
Re
lati
ve a
bu
nd
ance
Example of compromised instrument performance
10 20 30 40 50 60 70 80
40
30
20
10
0
42.1858.19 90.22
86.1546.10 56.29
59.8135.74
83.5729.6163.33 63.41 70.5054.44 75.9225.88
92.56 97.95 1
90 100
23.9111.10 20.39
Courtesy by MS BioWorks, Ann Arbor, MI
Good quality chromatogram (an instrument properly works)
Time(min)
Poor quality chromatogram (an instrument needs maintenance)
Peptide ionization and retention times are compromised
Detecting deterioration of an instrument performance in a timelymanner.
Re
lati
veab
un
dan
ceR
ela
tive
abu
nd
ance
40
30
20
10
0
40.09 51.15
60.3328.07 32.39 92.54 92.7866.58 74.10 82.28
12.00 25.9823.17
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6x5 peptide mixProduct concept
Isomer # Sequence MW M
1 LLSLGAGEFK 1072.67318 0.00
2 LLSLGAGEFK 1062.64598 10.03
3 LLSLGAGEFK 1055.62878 7.02
4 LLSLGAGEFK 1048.61158 7.02
5 LLSLGAGEFK 1041.59448 7.02
most hydrophilic peptide
MS
Peak
Inte
nsi
ty
LC Chromatogram C18 LC Gradient (increasing hydrophobicity)
most hydrophobic peptideIn
ten
sity
Peptide Retention Time
Linear Dynamic range
m/z
Six peptides. Each peptide is represented by five isotopologues mixed within linear concentration
dynamic range.
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A mixture of 6x5 = 30 peptides for completemonitoring of LC-MS/MS parameters
Each peptide has five chromatographically indistinguishable isotopologues, with abundances spanning four orders of magnitude. Bolded amino acids (in red) are uniformly labeled with stable 13C and 15N atoms.
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Beri et al. Analytical Chemistry 2015, 87, 11635−11640http://pubs.acs.org/doi/abs/10.1021/acs.analchem.5b04121
Mass spec products to be launched soonRapid Trypsin
2. Heat increases enzymatic activity.
1. Heat induces protein unfolding providing easy protease access to cleavage sites.
Heat12-18 hdigestion at 37oC
Heat advantages for proteolysis
Digested peptides Digested peptides
IgG digest IgG digest
Rapid Trypsin is a thermostable formulation of trypsin. It rapidly digests proteins at high temperature.
Conventional Protein Digestion
Denaturation
Reduction
Alkylation
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30 min digestion with no need for reduction and alkylation.
Rapid digestion with Rapid Trypsin
30 min at 70oC
Mass spec products to be launched soonRapid Trypsin
Rapid Trypsin is not for anytrypsin customer because ithas certain shortcomings.However, it perfectly fits the needs of the customers working in:
Drug discovery and pharmacokinetics fields
Proteomics
Clinical research12-18 hdigestion at 37oC
Digested peptides Digested peptides
IgG digest IgG digest
30 min digestion with no need for reduction and alkylation.
Rapid digestion with Rapid Trypsin
30 min at 70oC
Rapid Trypsin is a thermostable formulation of trypsin. It rapidly digests proteins at high temperature.
Conventional Protein Digestion
Denaturation
Reduction
Alkylation
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Mass spec products to be launched soonLow pH digestion kit for biotherapeutic protein characterization
This kit is designed for the most accurate characterization of biotherapeutic proteins with LC/MS and UV-HPLC.
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Mass spec products to be launched soon
Digestion
Digested peptides
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Denaturation
Reduction
Alkylation
Low pH digestion kit for biotherapeutic protein characterization
Digestion and other sample prep steps induce artificial posttranslational modifications including:
Deamidation
Disulfide bond scrambling
Oxidation
These modifications compromise characterization of biotherapeutic proteins.
Mass spec products to be launched soonLow pH digestion kit for biotherapeutic protein characterization
Digestion and other sample prep steps induce artificial posttranslational modifications including:
Deamidation
Disulfide bond scrambling
Oxidation
Digestion
Digested peptides
Denaturation
Reduction
Alkylation
Conventional digest
Low pH digest
peptide
Deamidation is eliminated
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Re
lati
ve
abu
nd
ance
These modifications compromise characterization of biotherapeutic proteins.
We have suppressed artificial PTMs by decreasing reaction pH from conventional alkaline range to acidic range (it was a very tricky thing to do!). This has significantly improved quality of biotherapeutic protein analysis.
Suppression of deamidation in RituximabGLEWIGAIYPGnGDTSYNQK peptide
Deamidated
Mass spec products to be launched soonRapid Trypsin and Low pH digestion kit
Both products have been extensively tested by customers. Customers are happy with the product performance and waiting for the product launch.
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