Tools to Improve Protein Sample
Preparation for Mass Spec
Sergei Saveliev, Ph.D.
R&D, Promega Corporation
Promega Corporation
1
Protein mass spec sample preparation
Common shortcomings
Protein extraction
Mass spec analysis
Poor protein extraction
from tissues
Inadequate instrument
performance monitoring
Protein fractionation
 Incomplete digestion
 Trypsin is not suitable
for analysis
 Poor peptide recovery
peptides
 Long and laborious
sample prep procedure
Protein digestion
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2
Enhanced Proteolysis with Trypsin/Lys-C Mix
 Minimized missed cleavages
 Increased tolerance to trypsin inhibiting agents
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3
Enhancing trypsin performance
Trypsin/Lys-C mix
Digestion
efficiency
 Highest digestion efficiency
 Tolerance to protease inhibiting agents
Trypsin Gold,
Mass Spec Grade
Trypsin, Sequencing Grade
Promega Corporation
 Higher digestion efficiency
 Higher purity
Overall good performance
4
What is the nature of incomplete proteolysis in
trypsin digests?
K
Overnight trypsin digest
of yeast protein extract
Trypsin cleavage sites
NNNNR NNNNK NNNN
Missed
cleavages
R
K
K
K K
K
22.2%
missed
cleavages
Missed K
18.6%
Missed R
3.6%
Majority of missed
cleavages occurs at
lysine sites.
2.6
4
K
Promega Corporation
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Solution: supplementing trypsin with Lys-C
Trypsin
Lys-C
NNNN(R/K) NNNN
NNNNK NNNN
Lysines are cleaved less efficiently.
Lysines are cleaved with high efficiency.
Lys-C is an ideal means to compensate for trypsin lysine cleavage
inefficiency.
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6
Enhanced proteolysis with Trypsin/Lys-C
Overnight digestion at 37°C
Trypsin digest
Missed K
18.6%
Missed R 3.6%
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3.6%
4%
7
Enhanced proteolysis with Trypsin/Lys-C
Overnight digestion at 37°C
Trypsin digest
Trypsin/Lys-C digest
Missed K
18.6%
Missed R 3.6%
3.6%
4%
Trypsin/Lys-C eliminates majority of missed cleavage sites.
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Study #1: Analysis of FFPE skin tissue
Sample prep is difficult due to extensive protein crosslinking in FFPE tissue.
Missed Cleavages, %
Trypsin
Identified Peptides
Trypsin
Identified Proteins
Trypsin/Lys-C
Trypsin
Trypsin/Lys-C
Trypsin/Lys-C
21.5%
705
887
8.5%
2.5 fold drop
24% increase
165
182
10% increase
Courtesy of Chris Adams, Stanford U
Trypsin/Lys-C increased number of identified peptides and
proteins in FFPE tissue.
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Study #2: Developing biomarker quantitative assay
for human plasma
Trypsin
digest
Trypsin/Lys-C
digest
Trypsin
digest
Trypsin/Lys-C
digest
674099
8976
145305
Peptide peak area
9139
SAP protein
Trypsin
digest
2207
Trypsin/Lys-C
Trypsin
digest
digest
3743
555
Trypsin/Lys-C
digest
1180
Courtesy of Matt Szapacs, GSK
Trypsin/Lys-C provided conditions for more accurate quantitation
of the targeted protein in plasma.
Promega Corporation
10
Study #3: Increased tolerance to trypsin inhibiting
agents
Digestion of yeast protein extract containing trypsin inhibiting agents
Missed (undigested) cleavage sites
Inhibitor
Protease inhibitor
cocktail, 1X
GuCl, 0.5 M
Protease
Identified proteins
Missed
cleavages
Trypsin
44.4%
Trypsin/Lys-C
21.5%
Trypsin
55.9%
Trypsin/Lys-C
24.6%
1495
1364
1252
Trypsin
13-20% increase
1204
Trypsin/
Lys-C
Trypsin
Inhibitor: protease
inhibitor cocktail
Trypsin/
Lys-C
Inhibitor: GuCl
Trypsin/Lys-C mix assures efficient proteolysis even if a protein
sample is contaminated with trypsin inhibiting agents.
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Advantages of Alternative Proteases
 Alternative cleavage specificity
 Activity under trypsin-inhibiting conditions
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Alternative cleavage specificity and
reaction conditions
Protease
Cleavage site
Property and application
Lys-C
NNNNK NNN
 Active under denaturing conditions
 Digest proteolytically resistant proteins
Glu-C
NNNNE NNN
Asp-N
NNNN DNNN
 Used as trypsin alternative if trypsin cleavage
sites have disadvantageous distribution
Arg-C
NNNNR NNN
(also cleave at K to a lesser degree)
 Analysis of histone post-translational
modifications
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
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Case study: digestion of membrane proteins
Too few tryptic cleavage sites
Tight folding prevents trypsin
access to cleavage sites
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.
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14
Increased protein sequence coverage with pepsin
and thermolysin
Bacteriorhodopsin
Coverage with trypsin
Sequence coverage, %
Coverage with pepsin
100
80
Pepsin
Thermolysin
60
Bacteriorhodopsin sequence coverage
40
20
Trypsin
0
Bacteriorhodopsin coverage was dramatically increased when
digested with thermolysin and pepsin.
Promega Corporation
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IdeS – Immunoglobulin Degrading Enzyme from
Streptococcus pyogenes
’
Fd’
IdeS
30 min.
digestion
LC
Fc/2
(+Glycans)
Mass spec
IdeS is an IgG-specific protease. It cleaves IgG at a unique site below the hinge.
IdeS offers a method for rapid and straightforward IgG analysis.
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Case study: Infliximab characterization
ximab_IdeSReduced_1ug_Promega_120313
ximab_IdeSReduced_1ug_Promega_120313
Fc/2
+Glycans
7.0e-2
7.0e-2
A214
5.0e-2
5.0e-2
4.0e-2
4.0e-2
Fc/2+Glycans
Infliximab_IdeSReduced_1ug_Promega_120313
5.
100
Fc/2
–C-term Lys
+Glycans
HPLC analysis of Infliximab digest with IdeS
%
6.0e-2
6.0e-2
2: Diode Array
214
0.1000Da
2:
Diode
Array
Range:
6.736e-2
214 0.1000Da
Range: 6.736e-2
Fd’
LC
3.0e-2
3.0e-2
2.0e-2
2.0e-2
1.0e-2
1.0e-2
%%
Intensity
100
100
0
6.00
0
6.00
7.00
7
7.00
8.00
8.00
10.00
10.00
11.00
11.00
12.00
12.00
13.00
13.00
Fc/2
+Glycans
-C term Lys
9.00
9.00
10.00
10.00
11.00
11.00
14.00
14.00
15.00
15.00
16.00
16.00
17.00
17.00
19.00
19.00
20.00
20.00
21.00
21.00
22.00
22.00
0
23.00
24.00
25000
25250
1: TOF24.00
MS ES+
23.00
TIC
1: TOF MS ES+
1.25e6
TIC
1.25e6
m
25500
25750
LC
Fd’
LC
12.00
12.00
13.00
13.00
Time
ACQUITY H-Class Bio, A 214, 2 Hz
Xevo G2 QTof, 500-4000m/z, 2 Hz
ACQUITY UPLC BEH300 C4, 1.7 µm, 300Å
2.1 x 150 mm, 0.2 mL/min
14.00
14.00
15.00
15.00
16.00
16.00
17.00
17.00
Infliximab_IdeSReduced_1ug_Promega_120313
1.
100
18.00
18.00
19.00
19.00
20.00
20.00
21.00
21.00
Mobile Phase A: 0.1% TFA, H2O
Mobile Phase B: 0.1% TFA, ACN
1 µg Reduced IdeS Digest (Chimeric IgG1)
Injection Volume: 0.67 µL
IdeS advantages for IgG characterization


18.00
18.00
%
Fc/2
+Glycans
0.0
6.00
7.00
8.00
9.00
0.0
ximab_IdeSReduced_1ug_Promega_120313
6.00
7.00
8.00
9.00
ximab_IdeSReduced_1ug_Promega_120313
22.00
22.00
23.00
23.00
22 min
0
Time
24.00
Time
23000
24.00
m
23250
23500
23750
Temp.: 80°C
5% to 33.3% ACN in 1 min,
then 33.3% to 40.3% ACN in 2
Ready separation of IgG fragments
Rapid analysis of major protein modifications
We have recently added IdeZ protease to our mass spec reagent portfolio.
IdeZ offers further improvement for IgG analysis. In contrast to IdeS, which preferentially
cleaves human antibodies, IdeZ also efficiently cleaves mouse antibodies.
Promega Corporation
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Protein Digestion in Gel with
MS Compatible Surfactant
 Increased peptide recovery
 In-gel digestion and peptide extraction
in a single 1h step
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18
In-gel protein digestion
Advantages and challenges
Advantages of SDS-PAGE protein fractionation
 Rapid removal of mass spec interfering impurities
 Efficient reduction of sample complexity
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In-gel protein digestion
Advantages and challenges
Shortcomings of in-gel protein digestion
 Inefficient peptide recovery from gel
 Extensive peptide loss due to adsorption to plasticware
 Lengthy and laborious procedure
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ProteaseMAX™ mass spec compatible surfactant
Degradation Products
ProteaseMAX™ Surfactant
Cleavable bonds
Hydrophobic tail
Zwitterionic head
+
Mass spec compatible anionic
surfactant
Degradation by
temperature or acid
LC/MS compatible compounds
Cleavable bonds
Self-degradable mass spec compatible surfactant
ProteaseMAX™ is designed to self-degrade over the course of mass spec
protein sample preparation into mass spec innocuous compounds.
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Improved peptide recovery
MALDI-TOF spectrum of HTR1A protein digested in gel
Peptides recovered with ProteaseMAX™
Peptides recovered in conventional digestion
Peptide
Increase in peptide recovery
with ProteaseMAX™, fold
AGGALCANGAVR
QGDDGAALEVIEVHR
EHLPLPSEAGPTPCAPASFER
1.45
2.06
1.80
Saveliev et al. Analytical Chemistry 2013, 85 (2), pp 907–914.
ProteaseMAX™ increases peptide recovery from gel.
Promega Corporation
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Minimized peptide adsorption to plasticware
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 plasticware.
Promega Corporation
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Enhanced analysis of a complex protein mixture
with ProteaseMAX™-assisted in-gel digestion
Gel-LC Analysis of Mouse Protein Extract
Courtesy of Dr. Chris Adams, Stanford U
ProteaseMAX™ increases number of peptide and protein
identifications in a cell extract digested in gel.
Promega Corporation
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Streamlined and rapid in-gel digestion with
ProteaseMAX™
Conventional In-gel Protein
Digestion
12-18 h
In-gel Protein Digestion with
ProteaseMAX™
Peptide extraction
Digestion/extraction step
Mass spec analysis
Mass spec analysis
(1.5 – 2 h)
Promega Corporation
(1 h)
Protein digestion
and peptide
extraction are
complete in a
single 1 h step.
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Protein Extraction from Tissues with
MS Compatible Surfactant
 MS compatible SDS analog for
tissue proteomics
 Efficient extraction of hydrophobic proteins
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Surfactant 3273 – MS compatible SDS analog for
tissue proteomics
Degradation Products
Surfactant 3273
Hydrophobic tail
Zwitterionic head
+
Cleavable bonds
Mass spec compatible anionic
surfactant
Degradation by
a strong acid
LC/MS compatible compounds
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.
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Improved protein extraction from pig liver with
surfactant 3273
Total extracted protein
Protein IDs in pig liver extracts
Total membrane protein
IDs in tissue extracts
3273
3273
RapiGest
SDS-PAGE
Control
Control
Chang et al., J. Proteome Res. 2015, 14 (3), pp 1587–1599.
3273 enhances protein extraction from animal tissues.
 Protein extraction efficiency is comparable to SDS.
 Number of recovered membrane proteins is significantly increased.
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Reference MS Protein Materials
 Validated test material for sample prep
optimization
 Standards for monitoring all key LC and
MS performance parameters
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MS-compatible whole cell protein extracts
Model proteomic material
Highly complex reference protein material for:

Mass spec instrument performance monitoring.

Sample preparation method development.
Features:
K562
human
cells
Yeast

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.
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
NL:
1.77E9
Base Peak
MS
msb16661re
p1
87.63
45.71
100
80
60
38.74
32.20 35.72
43.26
26.04
23.45
40
20
5.10
0
100
21.09
15.17 19.38
52.11
58.37
61.02
65.57
1st week
82.04
79.95
77.63
90.06
98.66
NL:
1.91E9
Base Peak
MS
msb16661re
p2
44.31
80
87.37
30.83
60
40
22.59
20
8.74
0
100
37.51
41.97
21.37
11.08 18.57
49.48 50.78
57.20
81.30 81.96
75.56 76.69
89.77
2nd week
97.62
NL:
1.83E9
Base Peak
MS
msb16661re
p3
38.22
45.10
80
87.91
22.92
60
31.60
35.20
25.48
40
48.88
20.61
8.10 14.88 18.94
20
0
0
10
20
30
3rd week
82.66
40
58.45
50
60
Time (min)
65.43
72.43
70
82.45
96.42 98.61
80
90
100
Detecting deterioration of instrument performance in a timely
manner.
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51.15
30
60.33
28.07 32.39
20
66.58
74.10
92.54 92.78
82.28
107.62
107.50
Example of compromised instrument performance
10
12.00
23.17 25.98
0
100
NL:
3.62E9
Base Peak
MS
msb17634b
49.83
90
80
70
Relative abundance
Good
quality chromatogram (an50.06
instrument properly works)
60
90.13
50
42.18
46.10
30
10 - 109.54
RT: 7.47
11.10
1000
56.29
35.74
20
10
20.39
90.22
58.19
40
25.88 29.61
23.91
54.44
86.15
59.81
63.33 63.41
83.57
70.50 75.92
92.56 97.95 100.21
52.87 53.02
20
30
40
50
60
Time (min)
90
70
80
90
100
109.06
80
89.84
NL:
1.46E9
Base Peak
MS
MSB17634A
70
Poor
quality chromatogram (an instrument needs maintenance)
108.24
Relative abundance
60
50
38.52
40
40.09
30
107.83
51.15
60.33
28.07 32.39
20
10
86.36
Peptide ionization
and89.92
retention times are compromised
45.54
12.00
66.58
74.10
82.28
92.54 92.78
107.62
107.50
23.17 25.98
0
100
Courtesy by MS BioWorks,
Ann Arbor, MI
NL:
3.62E9
Base Peak
MS
msb17634b
49.83
90
80
70
Detecting deterioration of instrument performance in a timely
manner.
50.06
60
90.13
50
42.18
46.10
30
56.29
35.74
20
25.88 29.61
23.91
54.44
Promega
11.10 Corporation
20.39
0
10
10
20
30
90.22
58.19
40
86.15
59.81
63.33 63.41
83.57
70.50 75.92
92.56 97.95 100.21
32
40
50
60
70
80
90
100
Convenient test material to optimize sample
preparation
Reliable test material for validating a phosphopeptide enrichment
resin.
Extract
Total
peptides
Phosphopeptides
Phosphopeptide
enrichment
Yeast
2502
1715
68%
Human
1768
1537
87%
Testing TiO2 resin for
phosphopeptide enrichment
Convenient model system for developing a peptide fractionation
method.
958 identified proteins
(14.5% proteome)
Yeast extract
W/o pre-fractionation
Promega Corporation
2446 identified proteins
(37% proteome)
Yeast extract
With high pH RP
pre-fractionation
Courtesy of Rebeca Pinhancos and
Nick Riley, UW-Madison
33
Quality control
Material quality
Extract
Human Yeast
Nonspecific cleavages
1%
2%
Deamidation spectra
4.9%
6.5%
Oxidation spectra
0.6%
1.3%
Carbamylated spectra
0.2%
0.6%
Missed tryptic cleavages
4.9%
4.8%
Spectrum match
65%
52%
Total spectra
26440
21264
Unique peptides
16375
12079
Identified proteins
2120
1637
 Virtual lack of protein fragmentation
 Low level of non-biological PTMs
 High number of protein IDs
1 µg injection, 2 h gradient, Q Exactive (Thermo)
The extracts pass strict QC control.
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Quality control
Lot-to-lot material reproducibility
Protein ID overlap
between extract lots
Courtesy of Stephanie Kaspar,
Bruker Daltonik GmbH
Lot-to-lot comparison of protein abundance
Courtesy of Brad Williams,
Waters Corporation
High lot-to-lot compositional and quantitative reproducibility.
Promega Corporation
35
6x5 peptide mix
Product concept
MS Peak Intensity
LC Chromatogram
most hydrophilic peptide
C18 LC Gradient (increasing
hydrophobicity)
most hydrophobic peptide
Peptide Retention Time
Intensity
Linear
Dynamic
range
Isomer #
1
2
3
4
5
Sequence
LLSLGAGEFK
LLSLGAGEFK
LLSLGAGEFK
LLSLGAGEFK
LLSLGAGEFK
MW
1072.67318
1062.64598
1055.62878
1048.61158
1041.59448
DM
0.00
10.03
7.02
7.02
7.02
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 complete
monitoring of LC-MS/MS parameters
Beri et al., Analytical Chemistry 2015, 87, 11635−11640
http://pubs.acs.org/doi/abs/10.1021/acs.analchem.5b04121
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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37
PreMiS software package
Report of all key LC and MS performance parameters
End-user collects data in
various formats
End-user generates a
report
LC Data Report
i. Data table
ii. XIC Analysis
Mass Spec
Analysis Report
MS Data report (DDA)
History Report
Multi-instrument
Report
 LC Parameters: Retention Time, Peak Height, Peak Width.
 MS Parameters: Mass accuracy, lowest detectable amount (sensitivity),
and linearity of detection.
 Thermo (.raw) and AB Sciex (.wiff) and .mzml formats.
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38
Thank you for attending our webinar!
Questions?
Now by chat…
…or later by email
Technical
Sergei Saveliev, R&D Senior Scientist
[email protected]
Promega Technical Services
[email protected]
Marketing
Promega Corporation
Gary Kobs, Sr. Global Product Manager
[email protected]
39