Overcoming Key Challenges of Protein Mass Spectrometry Sample Preparation Bottom-up proteomics is widely accepted as a primary method to characterize proteins. To ensure efficient protein analysis researchers must optimize key steps in the workflow to avoid potential pitfalls such as poor protein sample preparation and inconsistent LC-MS instrument performance. In this presentation, we will: • Investigate the cause of incomplete trypsin digestion and solution to this problem. • Discuss the advantage of alternative proteases for mass spec protein analysis. • Review the impact of mass spec compatible surfactants on protein digestion in gel and protein extraction from animal tissues. • Detail new reference mass spec protein and peptide materials designed to optimize protein sample preparation steps and monitor key instrument performance parameters. The presentation should prove valuable to any researcher using bottom-up proteomics, and who is concerned with improving protein mass spec sample preparation and mass spec instrument performance.

1. Overcoming key challenges of Protein Mass Spectrometry
Sample preparation
Mourad Ferhat, Ph.D
mourad.ferhat@promega.com

2. 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
The Feynman center cGMP facility (260,000 square feet = (24,155 m2), Madison for IVD
manufacturing

3. Drug Discovery Solutions by Promega
Bioassays
Cell Health In vivo imaging
Cell signaling
Ab characterization
Ab purification
ADMECell metabolism
Target engagement
Alternative Proteases
MS compatible Surfactants
Reference MS Protein Material
Trypsin digestion
Mass Spectrometry Reagents

4. Protein fractionation
Mass spec analysis
Protein mass spec sample preparation
peptides
Incomplete digestion
Trypsin is not suitable
foranalysis
Poor peptide recovery
Long and laborious
sample prep procedure
Poor protein extraction
fromtissues
Protein extraction
Inadequate instrument
performancemonitoring
Protein digestion
❶ Extraction
❷ Fractionation
❸ Digestion❹ Analysis

5. Enhanced Proteolysis
Trypsin, Rapid Trypsin, Trypsin/LysC Mix

6. 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
Trypsin Gold,
Mass Spec Grade
Trypsin/Lys-C mix

7. Nature of incomplete proteolysis in trypsin digests
Overnight trypsin digest
of yeast protein extract
22.2%
missed
cleavages
MissedR
3.6%
MissedK
18.6%
2.6
4
Majority of missed cleavages occurs
at lysine sites.
Missed
cleavages
Trypsin cleavage sites
NNNNR NNNNKNNNN
K
R
K
K
K K
K
K
R : Arginine
K : Lysine

8. Solution: supplementing trypsin with Lys-C
Lys-C is an ideal means to compensate for trypsin lysine cleavage
inefficiency.
Trypsin
NNNN(R/K) NNNN
Lysines are cleaved less efficiently.
NNNN K NNNN
Lysines are cleaved with high efficiency.
Lys-C

9. Enhanced proteolysis with Trypsin/Lys-C
Missed R 3.6%
MissedK
18.6%
Trypsin digest Trypsin/Lys-C digest
3.6%
4%
Trypsin/Lys-C eliminates majority of missed cleavage sites.
Overnight digestion at 37oC
Digestion of yeast protein extract containing trypsin inhibiting agents

10. Study #1: Analysis of FFPE skin tissue
21.5%
8.5%
Trypsin/Lys-C
Missed Cleavages, %
Trypsin
Trypsin
Identified Peptides
Trypsin/Lys-C
Identified Proteins
Trypsin
Trypsin/Lys-C
24% increase 10% increase2.5 fold drop
705
887
165 182
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.

11. Study #2: Developing biomarker quantitative assay for
human plasma
Courtesy of Matt Szapacs, GSK
674099
Trypsin/Lys-C
digest
9139
Trypsin/Lys-C
digest
Trypsin
digest
3743
Trypsin/Lys-C
digest
1180
Trypsin/Lys-C
digest
Trypsin
digestTrypsin
digest Peptide peak area
Trypsin
digest
SAP protein
145305
8976
2207 555
Trypsin/Lys-C provided conditions for more accurate quantitation
of the targeted protein in plasma.

12. Study #3: Increased tolerance to trypsin inhibiting
agents
Inhibitor:GuClInhibitor:protease
inhibitorcocktail
1252
1495
Trypsin/Lys-C mix assures efficient proteolysis even if a protein
sample is contaminated with trypsin inhibiting agents.
Inhibitor Protease Missed
cleavages
Protease inhibitor
cocktail, 1X
Trypsin 44.4%
Trypsin/Lys-C 21.5%
GuCl, 0.5 M
Trypsin 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

13. Rapid Trypsin : Trypsin digestion in as little as 60 min
2. Heat increases enzymatic
activity
1. Heat induces protein
unfolding providing easy
protease accessto cleavage
sites.
Heat advantages for proteolysis
Rapid Trypsin is a thermostable formulation of trypsin.
It rapidly digests proteins at high temperature.
 30 min digestion with no need for
reduction and alkylation.
 Rapid digestion with Rapid Trypsin
 30 min at70oC

14. Alternatives Proteases
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 cleavage
sites have disadvantageous distribution
Asp-N NNNN DNNN
Arg-C NNNNR NNN
(also cleave at K at a lesser degree)
 Analysis of histone posttranslational
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

15. 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
Toofew tryptic cleavage sites
Tight folding prevents trypsin
access to cleavage sites

16. 20
0
Bacteriorhodopsin coverage was dramatically increased when
digested with thermolysin and pepsin.
40
60
100
80
Pepsin
Thermolysin
Trypsin
Sequencecoverage,%
Bacteriorhodopsin
Coverage with trypsin
Coverage with pepsin
Bacteriorhodopsin sequence coverage
Increased protein sequence coverage with pepsin
and thermolysin

17. Biotherapeutics
Characterization
AccuMap™ Low pH digest, IdeS/IdeZ
proteases, Glycosidases

18. IdeS and IdeZ proteases
Antibody specific proteases

19. IdeS – Immunoglobulin Degrading Enzyme from
Streptococcus 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
IdeS advantage for IgG characterization
 Ready separation of IgG Fragments
 Rapid analysis of major protein modifications

20. IdeZ and IdeS protease cleavage specificity
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.

21. 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

22. Using EndoH and PNGase F to monitor protein
trafficking

23. AccuMAP™
Low pH digestion

24. Nonenzymatic PTMs induced during protein sample
preparation
LC-MS or UV-HPLC
Denaturation
Digestion
Reduction
Alkylation
Biotherapeutic protein
Digested
peptides
 Alkaline pH causes artificial PTMs
 Urea can cause protein carbamylation
 Deamidation
 Alkaline pH reagents causes artificial PTMs
 Excipients/impurities induce protein oxidation
 Incomplete digestion
 Baseline noise
 Long overnight incubation
 Poor reproducibility
 Disulfide bond
scrambling
 Oxidation
AccuMAP™ Low pH digest

25. Suppression of Artificial, Nonenzymatic PTMs with
AccuMAP™
Unmodifiedpeptide
Deamidated form
Scrambling issuppressed
46.13
47.44
46.08
Conventional digestion (pH 8)
Low pH digestion
HC319-LC134
scrambledbond
Mass= 497.7491;Z= 4
SGTASVVCLLNNFYPR
CK
39 40 41 42 43 44 45 46 47
Retentiontime(minutes)
48 49 50 51 52
0
50
100
0
50
100
68.5 69.5 71.5 72.5
70.93
70.83
70.78
70.68
70.45 70.62
70.16 70.36
71.06 71.31
71.46
71.58
71.77 72.15
69.0 70.0 70.5 71.0
Retentiontime(minutes)
72.0 73.0
B.
100
80 Conventional digestion (pH 8)
40
20
0
60
40
20
0
100
80
60 Low pH digestion
LC134
HC319
Deamidationissuppressed
A.
GLEWIGAIYPGNGDTSYNQK
 Deamidation suppression
 Disulfide bond scrambling suppression
Rituximab
(Rituxan™, Roche)
Panitumumab
(Vectibix®, Amgen)
+ AccuMAP™
+ AccuMAP™

26. Protein digestion in Gel
with MS Compatible surfactant
Protease Max™

27. 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

28. 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 digestion : Advantages and challenges

29. ProteaseMAX™ Surfactant
Cleavable bonds
Degradation by
temperature or acid
LC/MS compatible compounds
ZwitterionicheadHydrophobic tail
+
DegradationProducts
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 spec
protein sample preparation onto mass spec innocuous compounds.

30. Peptide Increase in peptide recovery with
ProteaseMAX™,fold
AGGALCANGAVR 1.45
QGDDGAALEVIEVHR 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.

31. Minimized peptide adsorption to plastic ware
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.

32. 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.

33. Conventional In-gel Protein
Digestion
Peptide extraction
(1.5 – 2 h)
Mass spec analysis Mass spec analysis
In-gel Protein Digestion with
ProteaseMAX™
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 with
ProteaseMAX™

34. Protein Extraction from Tissues
with MS Compatible surfactant
Surfactant 3273

35. Surfactant 3273 – MS compatible SDS analog for
tissue proteomics
Surfactant3273
Degradationby
a strong acid
Cleavable bonds
Mass spec compatible anionic
surfactant
DegradationProducts
Hydrophobic tail Zwitterionichead
+
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

36. Improved protein extraction from pig heart with
surfactant 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
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.

37. Reference MS
protein Materials
Yeast and Human Protein extracts
6 X 5 Peptide Mix

38. 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 extracts
Model proteomic material
K562 human
cells
Yeast
Reference Protein Materials address the critical question:
Do my mass spec instrument, reagents and method work properly?

39. 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
100
87.6345.71
38.74
23.45 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
87.37
30.83
37.51
41.9722.59 81.30 81.96
49.48 50.78 57.2021.37 89.7775.56 76.69
97.6218.5711.088.74
38.22
45.10
87.9122.92
31.60 82.66
35.2025.48 58.4548.88 82.4565.43
72.43
96.42 98.61
20.61
14.88 18.94
10 20
8.10
st1 week
nd2 week
3rd week
Detecting deterioration of an instrument performance in a timely
Manner (1 µg of human predigested protein extract)
Relativeabundance

40. Example of compromised instrument performance
10 20 30 40 50 60 70 80
40
30
20
10
0
42.18
58.19 90.22
86.15
46.10 56.29
59.8135.74
83.5729.61
63.33 63.41 70.5054.44 75.9225.88
92.56 97.95 1
90 100
23.91
11.10 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 timely
manner.
RelativeabundanceRelativeabundance
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

41. 6x5 peptide mix
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 peptideMSPeakIntensity
LC Chromatogram C18 LC Gradient (increasing
hydrophobicity)
most hydrophobic peptide
Intensity
Peptide Retention Time
Linear
Dynamic
range
m/z
 Six peptides.
 Each peptide is represented by five isotopologues mixed within linear concentration
dynamic range.

42. A mixture of 6x5 = 30 peptides for complete
monitoring 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.
Beri et al. Analytical Chemistry 2015, 87, 11635−11640
http://pubs.acs.org/doi/abs/10.1021/acs.analchem.5b04121

43. Mass Spectrometry solutions by Promega
Digestion
Trypsin
Trypsin Gold, Trypsin/Lys-C Mix, Sequencing grade,
immobilized Trypsin, Rapid Trypsin, AccuMAP™
Alternative proteases
Lys-C, Arg-C, Glu-C, Asp-N, Chymotrypsin, Pepsin,
ThermoLysin, Elastase
Glycosidases
PNGase, EndoH
Antibody specific protease
IdeZ IdeS protease
Extraction
Surfactant for in-gel protein
digestion
ProteaseMAX™ Surfactant for improved protein
digestion, extraction and solubilization
Protein extraction from Tissues
Surfactant 3273. That surfactant is designed for
efficient protein extraction from tissues and other
biological samples and solubilization of protein
pellets.
Instrument performance
monitoring
Protein extracts for LC/MS
Instrument
MS compatible Human or Yeast protein extracts for
instrument performance monitoring
Learn more about Promega solutions at:
www.promega.com/mass-spectrometry

44. Promega solutions for Proteomics
Expression
Expression Vectors
Mammalian expression vectors : regulated /constitutive
expression
Bacterial strains
KRX, BL21
Cell-free protein expression
Translation : Rabbit reticulocyte, Wheat Germ extract
Transcription and Translation : TnT® system (Reticulocyte,
Wheat Germ, Insect Cell), E.coli extracts
Purification
Affinity-based protein purification
His-Tagged proteins : HisLink™ (resin, spin column, well-
plates)
Biotinylated proteins : SoftLink™, PinPoint™
HaloTag® Fusion proteins : from E.coli & Mammalian cells
Magnetic beads (manual/automated)
MagneGST™, Magne™ HaloTag®, MagneHis™
Antibody purification
Magne™ Protein A/Magne™ Protein G
Labeling
Cell-free protein labeling
FluoroTect™ GreenLys, Transcend™
Biotinylated Lysine tRNA
HaloTag® Ligands
Coumarin, Alexa Fluor® 488/660, Oregon
Green®, TMR, R110
Processing/characterization
Membrane vesicles
Canine pancreatic microsomal membranes for signal
peptide cleavage and core glycosylation studies
Trypsin
Trypsin Gold, Trypsin/Lys-C Mix, Sequencing grade,
immobilized Trypsin
Alternative proteases
Lys-C, Arg-C, Glu-C, Asp-N, Chymotrypsin, Pepsin,
ThermoLysin, Elastase
Glycosidases
PNGase, EndoH, Fetuin
Surfactant
ProteaseMAX™ Surfactant for improved protein
digestion, extraction, solubilization compatible with
MS
Protein extracts for LC/MS
Instrument
MS compatible Human or Yeast protein extracts for
instrument performance monitoring
Antibody specific protease
IdeZ , IdeS
Interactions
Live cell PPIs
NanoBiT
NanoBRET
Drug discovery
Target engagement : NanoBRET TE
Target identification
Dectection/Capture
Western Blotting & ELISA
Substrates, Conjugated Secondary
antibodies
Protein Arrays
HaloLink™ Protein Array system