Overcoming Key Challenges of Protein Mass … fileOvercoming Key Challenges ... Detergents are generally avoided in mass spec field due to incompatibility with ... •Designed to self-degrade

©2013 Promega Corporation.

Overcoming Key Challenges of Protein Mass Spectrometry Sample Preparation with ProteaseMAX™ Surfactant Sergei Saveliev, Ph.D. Sr. Research Scientist II, Promega Corporation

In-gel protein digestion Improved protein

identification Streamlined procedure

A Mass Spec Compatible Surfactant for Multiple Protein Sample Preparation Applications

Protein extraction from cells and tissues High protein yield Improved recovery of

membrane proteins

Efficient digestion of tightly folded proteins Denatures proteins for

improved protease access

ProteaseMAX™ Surfactant

Protein solubilization Efficient re-solubilization of

precipitated proteins Solubilization of hydrophobic

proteins

2

ProteaseMAX™ Presentation Outline

• ProteaseMAX™ surfactant development

• ProteaseMAX™ surfactant advantage

o Enhanced and streamlined in-gel protein digestion

o Improved protein extraction from cells and tissues

o Efficient solubilization of protein pellets

o Aiding digestion of tightly folded proteins

• ProteaseMAX™ surfactant handling

• Summary

3

Detergents are generally incompatible with: o Reverse phase liquid chromatography o Mass spectrometry

Negative effects caused by detergents include: o Compromised peptide fractionation with liquid chromatography o High noise in mass spectra

Detergents and Mass Spec Protein Sample Preparation

Detergents (SDS, Triton X-100, etc.) have been used in protein sample preparation applications for decades.

Detergent advantages include:

Efficient extraction of proteins from cells, tissues and other biological samples Maintaining hydrophobic proteins in solution Re-solubilizing precipitated proteins

Detergents are generally avoided in mass spec field due to incompatibility with mass spec analysis and reverse phase liquid chromatography.

!

4

ProteaseMAX™ Surfactant A Mass Spectrometry Compatible Detergent

“Surfactant” = Surface acting agent ProteaseMAX™ Surfactant

• All the benefits of strong detergents such as SDS

• Compatibility with liquid chromatography and mass spectrometry

5

ProteaseMAX™ Surfactant Development An SDS-like Anionic Surfactant that’s MS Compatible

Ready degradation into mass spec compatible products

Efficient protein solubilization

Compatibility with trypsin and other proteases

Surfactant Library (over 40 compounds)

ProteaseMAX Surfactant Easily degraded Solubilizes membrane proteins at room temp Enables rapid protein digestion with trypsin

Searched the library for following criteria:

6

ProteaseMAX™ Surfactant Degradation Pathway

Cleavable bonds

ProteaseMAX™ Surfactant Anionic surfactant, 425.51 Da

Degradation by temperature or acid

Mass spec and reverse phase liquid chromatography compatible products

Zwitterionic head Hydrophobic tail

+

Stability of 1% Stock Solution

Time to 10% degradation

RT ~ 8 hours

+4 C ~12 days

-20 C > 3 years

Degradation Products

7

Temperature-induced Degradation

Time (min)

Surf

acta

nt

Co

nce

ntr

atio

n

0.00

0.01

0.02

0.03

0 10 20 30 40 50 60

Degradation of 0.025% ProteaseMAX™ at 50oC

7

ProteaseMAX™ Surfactant Self-degradation

Unusual, self-degradation feature • Designed to self-degrade over the course of mass spec protein sample preparation (during

protein digestion step)

Cleavable bonds

ProteaseMAX™ Surfactant Anionic surfactant, 425.51 Da

Degradation by temperature or acid

Mass spec and reverse phase liquid chromatography compatible products

Zwitterionic head Hydrophobic tail

+

Degradation Products

8



proteins





membrane proteins




9

In-gel Protein Digestion Benefits and Challenges

Step 1 Prepare protein extract

Step 2 Fractionate proteins in

gel

Step 6 Analyze by mass spec

Step 3 Cut the protein

band from gel

Step 4 Digest protein

in gel (with trypsin)

Step 5 Extract

peptides

Protein fractionation by SDS-PAGE is a very popular approach in mass spec field:

SDS-PAGE efficiently removes mass spec interfering impurities from a protein sample

Gel fractionation reduces complexity of biological samples

10

In-gel protein digestion challenges:

Extensive peptide loss decreases efficiency of mass spec analysis o Protein coverage is decreased o Low abundant proteins are difficult to identify

Lengthy and laborious procedure

Step 1 Prepare protein extract

Step 2 Fractionate proteins in

gel

Step 6 Analyze by mass spec

Step 3 Cut the protein

band from gel

Step 4 Digest protein

in gel (with trypsin)

Step 5 Extract

peptides

In-gel Protein Digestion Benefits and Challenges

11

Systematic study of ProteaseMAX™ effects on in-gel protein digestion.

ProteaseMAX™-assisted In-gel Protein Digestion

Analytical Chemistry 2013, 85: 907-914

12

ProteaseMAX™-assisted In-gel Protein Digestion Improved Protein Identification

MALDI-TOF Analysis of In-gel Digested BSA

Enhanced In-gel Protein Digestion

• Increased protein sequence coverage and probability of protein identification

The digests were analyzed with AB 4800 MALDI-TOF/TOF

13

ProteaseMAX™-assisted In-gel Digestion Rapid Digestion

MALDI-TOF Analysis of In-gel Digested BSA

Rapid In-gel Protein Digestion

• 4 hours at 37°C • 1 hour at 50°C

The digests were analyzed with AB 4800 MALDI-TOF/TOF

14

The digests were analyzed with AB 4800 MALDI-TOF/TOF.

MALDI-TOF Spectra of In-gel Digested BSA

Conventional Overnight Digestion

1hr Digestion with ProteaseMAX™

Seq. coverage: 47% Mascot score: 498

Seq. coverage: 64% Mascot score: 557

Improved recovery of larger peptides

ProteaseMAX™-assisted In-gel Digestion Improved Recovery of Larger Peptides

15

MALDI-TOF Analysis of HTR1A Protein Digested with and without ProteaseMAX™

Sequence Peptide mass

Surfactant-induced increase in peptide recovery, fold

Light isotope (12C) version Heavy isotope (13C) version

AGGALCANGAVR 1116.6 Da 1122.6 Da 1.45+/-0.28

QGDDGAALEVIEVHR 1608.8 Da 1614.8 Da 2.06+/-0.75

EHLPLPSEAGPTPCAPASFER 2263.1 Da 2269.1 Da 1.80+/-0.34

Surfactant-induced Increase in Peptide Recovery

• Heavy (13C-labeled) HTR1A protein in-gel digested with trypsin + ProteaseMAX™

• Light (12C-labeled) HTR1A protein in-gel digested with trypsin only

Extracted peptides were mixed and analyzed with MALDI-TOF

ProteaseMAX™-assisted In-gel Digestion Improved Peptide Recovery

Note higher peptide intensity with ProteaseMAX™

16

• Adsorption to plasticware is the major cause of peptide loss in in-gel digestion

• ProteaseMAX™ minimizes this adsorption

• Probably, the absorption is prevented by one of the degradation products, the hydrophobic tail, which has non-ionic surfactant properties.

Reduced Peptide Adsorption to Reaction Tube

ProteaseMAX™-assisted In-gel Digestion Minimized Peptide Loss Due to Adsorption to Plastic

17

ProteaseMAX™-assisted In-gel Digestion Protocol 1 Hour Digestion and Simultaneous Extraction

1 hour digestion

Mass spec analysis

In-gel Protein Digestion with ProteaseMAX™

• Simultaneous protein digestion and peptide extraction in 1h

18

Comparison of Sequence Coverage for Proteins Digested In-gel with and without ProteaseMAX™

Protocol Validation Improved Protein Coverage

19

Identification of Low Abundance Proteins Digested In-gel with and without ProteaseMAX™

Protocol Validation Improved Identification of Low Abundance Proteins

1. Proteins were loaded in gel at low nanogram quantities 2. In-gel digested with trypsin +/- ProteaseMAX™ 3. Analyzed by MALDI-TOF

ProteaseMAX™ increased number of identified low abundance proteins from 10 to 19

ProteaseMAX™ improved the MASCOT score for 18/19 identified proteins

20

Protocol Validation Improved Protein Identification in Complex Mixture

Gel-LC Analysis of Mouse Protein Extract

Courtesy by Dr. Chris Adams, Stanford U

Protocol Resolve membrane protein extract from mouse heart by SDS-PAGE

Excise lanes onto 9 fractions

Digest for 1hr with Trypsin+ ProteaseMAX™ or overnight according to a conventional trypsin protocol

Analyze with LCQ Deca XP+/Eksigent 2D Nano LC

21

Gel-LC Analysis of Mouse Protein Extract – the Results

Courtesy by Dr. Chris Adams, Stanford U

Greater number of peptide and protein identifications

Protocol Validation Improved Protein Identification in Complex Mixture

22

Conventional In-gel Protein Digestion

ProteaseMAX™-assisted In-gel Digestion Enhanced Analysis and Streamlined Protocol

Overnight digestion

Peptide extraction (1.5 - 2hr)

Mass spec analysis

1 hour digestion

Mass spec analysis

In-gel Protein Digestion with ProteaseMAX™

• Simultaneous protein digestion and peptide extraction

• Rapid protocol (1h versus overnight)

• Improved peptide recovery, protein ID and sequence coverage

15hr

23

ProteaseMAX™-assisted In-gel Digestion Protocol is Readily Available in the Technical Bulletin

Technical Bulletin available online at: http://www.promega.com/~/media/Files/Resources/Protocols/Technical%20Bulletins/101/ProteaseMAX%20Surfactant%20Trypsin%20Enhancer.pdf

24

http://www.promega.com/~/media/Files/Resources/Protocols/Technical Bulletins/101/ProteaseMAX Surfactant Trypsin Enhancer.pdf











proteins






membrane proteins

25

Enhanced Cell Lysis and Protein Extraction with ProteaseMAX™ from Human A375 Cells

Comparison of ProteaseMAX™, Na Deoxycholate and Urea for Cell Lysis and Protein Extraction

One critical factor to achieving the highest proteome coverage is efficient cell lysis & protein extraction.

A375 human cells

Cell lysis with one of the reagents: Urea Deoxycholate ProteaseMAX™

Protein extraction

Trypsin digestion

Clean-up

LC Easy Spray Data analysis

“Rapid and deep human proteome analysis by single-dimension shotgun proteomics”. Pirmoradian et al. MCP Papers in Press. Published on July 22, 2013

26

ProteaseMAX™ improved protein recovery

50% coverage of the expressed human proteome

Enhanced Cell Lysis and Protein Extraction with ProteaseMAX™ from Human A375 Cells

Most improvement for membrane, nuclear, cytoplasmic and cytosolic proteins

27

Extraction and Analysis of Rat Brain Tissue

Solubilize rat brain homogenate pellet in ProteaseMAX™/Urea or, alternatively, Invitrosol™*/Urea

Digest with trypsin

Fractionate peptides with MudPIT

LC-MS/MS

*Invitrosol™ is a mass spec-compatible surfactant by Life Technologies

ProteaseMAX™/Urea mix:

• 15µl 8M urea and 20µl 0.2% ProteaseMAX™

Courtesy of Dr. Dan McClatchy

Improved Protein Extraction from Rat Brain Tissue with ProteaseMAX™ + Urea

28

Peptides and Proteins Identified in the Rat Brain Extracts

Invitrosol™/Urea ProteaseMAX™/Urea Increase with ProteaseMAX™

20.4% 42.8% Two fold

Protein Sequence Coverage for Glutamate Receptor

Total Identified Proteins Identified Membrane Proteins

Invitrosol™/Urea

1699 2251

535 694

ProteaseMAX/Urea

Invitrosol™/Urea

ProteaseMAX/Urea

Courtesy of Dr. Dan McClatchy

ProteaseMAX™/Urea mix create an efficient protein extractor/solubilizer

Improved Protein Extraction from Rat Brain Tissue with ProteaseMAX™ + Urea

29

Efficient digestion of tightly folded proteins Deanatures proteins for






membrane proteins




proteins

30

Re-solubilizing Protein Pellets

• Acetone precipitation is a popular method to: • Remove mass spec interfering impurities from proteins • Concentrate dilute protein samples

• After precipitation, protein pellets are very difficult to re-solubilize

• Growing numbers of laboratories are now using a mix of ProteaseMAX™ and Urea as a pellet solubilizer

31

ProteaseMAX™ in Combination with Urea Creates an Efficient Protein Pellet Solubilizer

Precipitate proteins with cold acetone

Removes mass spec interfering agents

Dissolve protein pellet with ProteaseMAX™/Urea mix

Digest with trypsin and analyze with mass spectrometry

Protein Resolubilization Protocol

Pearson et al. Regulation of H2O2 Stress-responsive Genes through a Novel Transcription Factor in the Protozoan Pathogen Entamoeba histolytica. JBC 2013, 288, 4462-4474

Schauer et al. Mass Spectrometry Contamination from Tinuvin 770, a Common Additive in Laboratory Plastics. J. Biomol. Techn. 2013, 24, 57-61

Bisson et al. Upregulation of the Phthiocerol Dimycocerosate Biosynthetic Pathway by Rifampin-Resistant, rpoB Mutant Mycobacterium tuberculosis. J. Bacteriol. 2012, 194, 6441-6452

Rothbard et al. Therapeutic Effects of Systemic Administration of Chaperone B-Crystallin Associated with Binding Proinflammatory Plasma Proteins. JBC 2012, 287, 9708-9721

Current Publications Using ProteaseMAX™/Urea to Solubilize

Acetone-Precipitated Proteins

Re-solubilizes difficult protein pellet

32

Hydrophobic Protein Solubilization

HPLC Chromatograms of Membrane Protein Bacteriorhodopsin Chymotrypsin Digests

Bacteriorhodopsin was not solubilized in an aqueous solution and not digested by chymotrypsin

Chymotrypsin Only in Aqueous Solution

Chymotrypsin Plus ProteaseMAX™ in Aqueous Solution

Peptides Bacteriorhodopsin was digested by chymotrypsin due to solubilization by ProteaseMAX™

Retention time, min

Retention Time, min

ProteaseMAX™ Surfactant: is an efficient solubilizer of hydrophobic proteins effectively solubilizes these difficult proteins in a few minutes at room temp

33



proteins





membrane proteins




34

Enhancing Trypsin Digestion of Tightly Folded Proteins

HPLC Chromatograms of Myoglobin Trypsin Digests

Trypsin Only

Trypsin Plus ProteaseMAX™ Myglobin Peptides

Intact Myoglobin Myoglobin tolerated digestion (myoglobin is a proteolytically resistant protein)

Myoglobin was completely digested by Trypsin in 30 minutes

ProteaseMAX™ Surfactant helps trypsin digest difficult proteins

Retention time

Retention time

35

Digestion Mechanism

Inaccessible sites

Add ProteaseMAX™

Access by Trypsin

Denatures the protein making internal sites accessible to trypsin

Tight folding inhibits trypsin digestion

Trypsin rapidly digests the protein

Enhancing Trypsin Digestion of Tightly Folded Proteins by Relaxing Protein Structure

36

Tested/compatible proteases:

• Lys-C

• Glu-C

• Chymotrypsin

ProteaseMAX™ is Compatible with Other Proteases

37

Handling ProteaseMAX™ Surfactant

Hydrophobic portion of degraded ProteaseMAX™

Peptides

HPLC Chromatogram of ProteaseMAX™-assisted Protein Digest

• Although ProteaseMAX™ degradation products do not generally interfere with liquid chromatography and mass spectrometry, it is recommended to remove these products prior to analysis.

• Potential negative effects of the degradation products if the surfactant is present in excess:

• Peptide precipitation • Column clogging

Note: Hydrophobic degradation

product elutes very late in the LC gradient • No effect on column

binding capacity or peptide elution time

Zwitterionic head is not retained on column

38

Handling ProteaseMAX™ Surfactant Removal of Degradation Products

Degraded ProteaseMAX™

Before Centrifugation

After Centrifugation Degraded ProteaseMAX™

Is removed

Degradation products are readily removed by • Centrifugation • Solid phase extraction

Peptides

Peptides

39

Handling ProteaseMAX™ Surfactant Carry-over Removal

Base Peak Chromatogram of ProteaseMAX™-assisted In-gel Digest

• Degraded ProteaseMAX™ may carry-over to next LC run (along with hydrophobic peptides, fatty acids and other compounds from a biological sample)

• A blank LC run is recommended to remove the degraded surfactant

090129_promega_55mw_p_02 1/29/2009 11:46:40 PM

RT: 14.72 - 80.00

15 20 25 30 35 40 45 50 55 60 65 70 75

Time (min)

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bu

nd

an

ce

73.10

41.9831.37

41.78

35.6328.6045.4733.83

36.67

50.6443.5924.38 45.60

56.3624.58 37.94 69.1348.3150.84 69.26

74.4120.0963.74 66.8163.20 75.4421.5616.29

72.92

72.78

41.67

31.42

45.14

35.53 36.4431.02

33.8750.05

55.9724.2737.63 68.5256.11

47.9950.40

62.68 75.2419.55 72.2463.2260.85 77.5117.35

NL:

5.06E9

Base Peak

MS

090129_Pr

omega_55

MW_P_01

NL:

5.08E9

Base Peak

MS

090129_pr

omega_55

mw_p_02Peptides Degraded ProteaseMAX

40

Handling ProteaseMAX™ Surfactant LC Column Carry-over Removal

A Simple blank LC Run Removes Degradation Product carry-over

No detectable signal decrease or deterioration in chromatographic profiles have been observed after >100 LC-MS runs with ProteaseMAX™ Surfactant-containing samples

41





ProteaseMAX™ Aids Mass Spec Protein Sample Preparation in Multiple Ways


membrane proteins

ProteaseMax™ Surfactant



proteins

42

Thank You for Attending Our Webinar!

Technical Sergei Saveliev, R&D Senior Scientist [email protected] Promega Technical Services [email protected]

Marketing Gary Kobs, Strategic Marketing Manager [email protected]

Questions?

Now by chat…

…or later by email

43

mailto:[email protected]



Overcoming Key Challenges of Protein Mass … fileOvercoming Key Challenges ... Detergents are generally avoided in mass spec field due to incompatibility with ... •Designed to self-degrade

Documents