overcoming key challenges of protein mass … fileovercoming key challenges ... detergents are...
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©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
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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
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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.
!
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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
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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:
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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
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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
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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
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Protein solubilization Efficient re-solubilization of
precipitated proteins Solubilization of hydrophobic
proteins
Efficient digestion of tightly folded proteins Denatures proteins for
improved protease access
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
ProteaseMAX™ Surfactant
In-gel protein digestion Improved protein
identification Streamlined procedure
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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
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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
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Systematic study of ProteaseMAX™ effects on in-gel protein digestion.
ProteaseMAX™-assisted In-gel Protein Digestion
Analytical Chemistry 2013, 85: 907-914
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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
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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
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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
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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™
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• 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
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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
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Comparison of Sequence Coverage for Proteins Digested In-gel with and without ProteaseMAX™
Protocol Validation Improved Protein Coverage
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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
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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
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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
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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
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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
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Efficient digestion of tightly folded proteins Denatures proteins for
improved protease access
Protein solubilization Efficient re-solubilization of
precipitated proteins Solubilization of hydrophobic
proteins
In-gel protein digestion Improved protein
identification Streamlined procedure
A Mass Spec Compatible Surfactant for Multiple Protein Sample Preparation Applications
ProteaseMAX™ Surfactant
Protein extraction from cells and tissues High protein yield Improved recovery of
membrane proteins
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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
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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
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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
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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
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Efficient digestion of tightly folded proteins Deanatures proteins for
improved protease access
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
ProteaseMAX™ Surfactant
Protein solubilization Efficient re-solubilization of
precipitated proteins Solubilization of hydrophobic
proteins
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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
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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
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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
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Protein solubilization Efficient re-solubilization of
precipitated proteins Solubilization of hydrophobic
proteins
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
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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
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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
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Tested/compatible proteases:
• Lys-C
• Glu-C
• Chymotrypsin
ProteaseMAX™ is Compatible with Other Proteases
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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
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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
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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
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Efficient digestion of tightly folded proteins Denatures proteins for
improved protease access
In-gel protein digestion Improved protein
identification Streamlined procedure
ProteaseMAX™ Aids Mass Spec Protein Sample Preparation in Multiple Ways
Protein extraction from cells and tissues High protein yield Improved recovery of
membrane proteins
ProteaseMax™ Surfactant
Protein solubilization Efficient re-solubilization of
precipitated proteins Solubilization of hydrophobic
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
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