Overcoming key challenges of Protein Mass Spectrometry

Sample preparation

Mourad Ferhat, Ph.D

mourad.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 ShanghaiThe Feynman center cGMP facility (260,000 square feet = (24,155 m2), Madison for IVD

manufacturing

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

Protein fractionation

Mass spec analysis

Protein mass spec sample preparation

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

❶ Extraction❷ Fractionation

❸ Digestion❹ Analysis

Enhanced Proteolysis

Trypsin, Rapid Trypsin, Trypsin/LysC Mix

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

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 missed cleavages occursat lysine sites.

Missed cleavages

Trypsin cleavage sites

NNNNR NNNNKNNNN

K

RK

K

K K

KK

R : Arginine

K : Lysine

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

Enhanced proteolysis with Trypsin/Lys-C

Missed R 3.6%

Missed K 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

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

165 182

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

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.

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

Rapid Trypsin : Trypsin digestion in as little as 60 min

2. Heat increases enzymaticactivity

1. Heat induces protein unfolding providing easy protease access to cleavagesites.

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 at 70oC

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 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

20

0

Bacteriorhodopsin coverage was dramatically increased whendigested with thermolysin and pepsin.

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 pepsin

and thermolysin

Biotherapeutics

Characterization

AccuMap™ Low pH digest, IdeS/IdeZ

proteases, Glycosidases

IdeS and IdeZ proteases

Antibody specific proteases

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

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.

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

Using EndoH and PNGase F to monitor protein

trafficking

AccuMAP™

Low pH digestion

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

Suppression of Artificial, Nonenzymatic PTMs with

AccuMAP™

Unmodifiedpeptide

Deamidated form

Scrambling is suppressed

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

Retention time(minutes)

48 49 50 51 52

0

50

100

0

50

100

68.5 69.5 71.5 72.5

70.93

70.8370.78

70.6870.45 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

Retention time(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

Deamidation is suppressed

A.

GLEWIGAIYPGNGDTSYNQK

Deamidation suppression

Disulfide bond scrambling suppression

Rituximab

(Rituxan™, Roche)

Panitumumab

(Vectibix®, Amgen)

+ AccuMAP™

+ AccuMAP™

Protein digestion in Gel

with MS Compatible surfactant

Protease Max™

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

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

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.

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.

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.

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 proteinidentifications in a cell extract digested in gel.

Conventional In-gel Protein Digestion

Peptide extraction(1.5 – 2 h)

Mass spec analysis Mass spec analysis

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 with

ProteaseMAX™

Protein Extraction from Tissues

with MS Compatible surfactant

Surfactant 3273

Surfactant 3273 – MS compatible SDS analog for

tissue 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

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 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.

Reference MS

protein Materials

Yeast and Human Protein extracts

6 X 5 Peptide Mix

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?

Mass spec instrument performance monitoringRT: 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

st1 week

nd2 week

3rd week

Detecting deterioration of an instrument performance in a timelyManner (1 µg of human predigested protein extract)

Rela

tive a

bu

nd

an

ce

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

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 peptideM

SPe

akIn

ten

sity

LC Chromatogram C18 LC Gradient (increasing hydrophobicity)

most hydrophobic peptide

Inte

nsi

tyPeptide Retention Time

Linear Dynamic range

m/z

Six peptides. Each peptide is represented by five isotopologues mixed within linear concentration

dynamic range.

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, 11635−11640http://pubs.acs.org/doi/abs/10.1021/acs.analchem.5b04121

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

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