Center for Mass Spectrometry and Proteomics | Phone | (612)625 … · 2019-03-29 · Center for...
Transcript of Center for Mass Spectrometry and Proteomics | Phone | (612)625 … · 2019-03-29 · Center for...
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
Bottom Up Proteomics
Top Down Proteomics
Proteins
Proteolytic
Digestion
HPLC
or SPE
Proteins Peptides Mass Spec.
FT-ICR Mass Spec.
Analysis
Analysis
HPLC, Gelfree, CE
adpated from http://www.piercenet.com/method/sample-preparation-mass-spectrometry
Two Main Approaches to Proteomics
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
Sample preparation is one of the most important aspects of any
successful proteomics experiment!
Simple protein ID
Differentially expressed proteins (ITRAQ, SILAC, label free or targeted)
Post-translational modifications (PTM)
Cytoplasmic, membrane, nuclear, mitochondrial, etc.
Immunoprecipitation/binding partners
2. What do I hope to analyze?
Questions you should ask before proteomics sample prep:
1. How should I collect the samples?
Consistency, consistency…
...and more consistency
Sampling for biological variation
Document all steps from sample collection thru prep – Good lab notes!
Mass Spec.
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
3. How do I best extract my proteins of interest?
In-gel: SDS-PAGE (1D or 2D)
In-solution
Enrichment/affinity strategies
Depletion of high abundant proteins
4. Is the extraction buffer compatible with mass spectrometry? If not, how do I get rid of problematic buffer components?
Solid Phase Extraction (SPE), detergent removal spin-column
Cloud point extraction of non-ionic detergents
Ethyl acetate extraction of detergents
Protein precipitation
Dialysis, molecular weight cut-off spin filter
Questions before starting prep (continued) :
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
Image: http://www.piercenet.com/method/sample-preparation-mass-spectrometry
Overview: Sample Prep to Mass Spectrometry Work Flow
2DE
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
Lysate Preparation/Protein Extraction
Solid Tissue and cell culture samples Biological Fluids, Media, other solution preps (i.e. IP’s)
Disruption
sonication
pressure cycling technology bead beater/homogenizers
Enzymatic Heat, Freeze/thaw +
Extraction Buffer (denature proteins)
Chaotropes(Disorder maker)
Denaturing Buffer
Detergents Urea, thiourea, guanidine HCl, salts(KCl, NaCl, etc.), MeOH, acetonitrile
Ionic (SDS), nonionic (NP40, triton x-100), zwitterionic (CHAPS)
MW Spin filter Dialysis Precipitation
Buffer reagent – pH considerations Tris, HEPES, ammonium bicarbonate, PBS, TEAB
Reducing reagents Dithiothreitol (DTT), TCEP (tris(2-carboxyethyl)phosphine)
Depletion of high abundant proteins typically done before digestion Enrichment of post-translational modifications typically done after digestion
Common Extraction Buffers: 4% SDS, 100mM Tris pH 7.6, 100mM DTT 7M urea, 2M thiourea, 0.4M TEAB pH8.5, 20% acetonitrile, 4mM TCEP
freeze + mortar & pestle
Centrifuge insoluble material out & aspirate supernatant (protein extract)
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
Disruption with the Barocycler® NEP2320
Maximizing Protein Extraction from Tissue
Pressure Cycling Technology Sample Preparation System
(PCT SPS)
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
…the Power of PCT
Pressure Cycling Technology (PCT) uses cycles of hydrostatic pressure between ambient and ultra high levels allowing for a high degree of speed, reproducibility, and convenience.
1 PSI input produces 440 PSI output resulting in a top pressure of 35 kPSI.
Better extraction from various sample types and quicker enzymatic treatments (proteolytic cleavage, deglycosylation, etc.)
-5000
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5000
10000
15000
20000
25000
30000
35000
3:56:10 3:56:53 3:57:36 3:58:19 3:59:02 3:59:46
Pressure Biosciences, Inc.
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
Pressure Compresses Lipids Beyond
Equilibrium
Hydrostatic Pressure
Applied
Alexander Lazarev, Pressure BioSciences (2006), CHI G.O.T. Summit 2006, Boston, MA
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
Rapid De-pressurization Causes Membranes
and Micelles to Disintegrate
Hydrostatic Pressure
Rapidly Released
Alexander Lazarev, Pressure BioSciences (2006), CHI G.O.T. Summit 2006, Boston, MA
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
PCT = pressure cycling technology, 10cycles, 20sec. 35kpsi, 20sec ambient press.
GG = ground glass dounce homogenizer
Rat Liver Preps for 2DE, H. Ringham et al. Electrophoresis 2007, 28, 1022–1024
500 ug loaded on
each gel
image: www.chemie.unihamburg.de/ bc/hahn/equipment/index_e.html
image: www.opsdiagnostics.com
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
-/+ Barocycler Prep & Digestion mouse liver tissue
Treatment ABI 4800 Protein ID’s
Thermo LTQ Protein ID’s
Std. Lysis/Std. Digest 115 (99%Protein Pilot)
203 (95%Protein Pilot)
118 (≥3peptides 99%,
Scaffold)
Std. Lysis/PCT Digest
NR 219
PCT Lysis/Std. Digest
275
471
264
PCT Lysis = 30 cycles @ 37C, 50sec.
35kpsi, 10sec. Ambient press.
PCT Digest = 60 cycles @ 37C,
50sec. 20kpsi, 10sec. Ambient press.
Std. Lysis = mortar & pestle + sonication
Std. Digest = overnight(16hrs.)
Lysis Buffer = 8M urea, 0.2%SDS, 0.5M TEAB, 5mM TCEP
Std. Digest = reduce, alkylate, dilute sample to 2M urea, add trypsin in 1:25 ratio, incubate overnight @ 37C
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
Biological Fluid Prep Example - Urine
Urine Sample
MW Cut-off Spin Filter
Recover sample
Vacuum Centrifuge to Dryness
Dialyze
Resuspend with Solubilization buffer
Proteolytic
Digestion
HPLC
or SPE
Mass Spec.
Protein Concentration Assay (Bradford or BCA)
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
http://journal.frontiersin.org/Journal/10.3389/fpls.2013.00052/full
“When You Come to a Fork in the Road, Take It!” –Yogi Berra
Two main sample prep paths to take...choose wisely!
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
In-gel Proteolytic Digestion
workflow picture: www.piercenet.com/method/sample-preparation-mass-spectrometry Adapted from: www.cshprotocols.cshlp.org
+ trypsin, 37°C
R K
R
DTT or TCEP + heat
iodoacetamide (+57 mass shift on Cys) methyl methanethiosulfonate (+46 on Cys)
: 50/50 acetonitrile/water, 0.1% TFA or FA 75/25 acetonitrile/water, 0.1% TFA or FA pool digest and extracts for same sample dry down in vacuum centrifuge for SPE or mass spec.
Incubate trypsin digest overnight @ 37°C
ammonium bicarb./acetonitrile washes dry with 100% acetonitile – no speedvac
5ng/ul
gel cutting pictures: http://sites.psu.edu/msproteomics/category/sample-prep/in-gel-digestion-tutorial/
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
In-Solution Proteolytic Digestion
*Protein Extract
* Peptides
Dilute buffer components that interfere with proteolytic enzyme: urea < 2M, thiourea < 1M SDS < 0.1%
Digest: Add trypsin (proteolytic enzyme) in 1:35 to 1:100 enzyme to total protein ratio
Incubate overnight (12-16hrs) @ 37°C
Freeze digest at -80°C and dry down for SPE of peptides
*Consider if buffer components can be cleaned up with SPE or HPLC step before mass spec., otherwise need to rethink buffer
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
Reference: JR Wiśniewski, M Mann, et.al, Nature Methods 6, 359 - 362 (2009)
Peptides Load Lysate
Proteins Retained
Adapted from www.piercenet.com
Buffer Exchange
Digest
Filter Aided Sample Prep - FASP
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
Many different proteolytic enzymes – choose based on your application
www.promega.com
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
Solid Phase Extraction (SPE)/Clean-up of Peptides
Peptides + Digest Buffers
Only Buffer & Salts other anionic hydrophobic
contaminants
Nonionic/Zwitterionic/ionic Detergents (triton-x100, CHAPS, NP40, SDS, etc.
C18 material
C18 resin image: www.lamondlab.com tips & Empore image: www.sigmaaldrich.com
MCX - Mixed Mode Cation Exchange
2 punches
Sodium Dodecyl Sulfate (SDS)
image: biotage.phosdev.se ProteoSpin: http://norgenbiotek.com/display-product.php?ID=7 cartridge images: www.perkinelmer.com
Pierce detergent removal: http://www.fishersci.com
Cloud Point Extraction – only for nonionic detergents (J. Proteome Res., 2010, 9 (8), pp 3903–3911)
Ethyl acetate precipitation of detergents (YG Yeung, Curr Protoc Protein Sci. Feb 2010; CHAPTER: Unit–16.12.)
Detergent removal spin columns
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
Lysate Preparation/Protein Extraction
Solid Tissue and cell culture samples Biological Fluids, Media, other solution preps (i.e. IP’s)
Disruption
sonication
pressure cycling technology bead beater/homogenizers
Enzymatic Heat, Freeze/thaw +
Extraction Buffer (denature proteins)
Chaotropes(Disorder maker)
Denaturing Buffer
Detergents Urea, thiourea, guanidine HCl, salts(KCl, NaCl, etc.), MeOH, acetonitrile
Anionic (SDS), nonionic (NP40, triton x-100), zwitterionic (CHAPS)
MW Spin filter Dialysis Precipitation
Buffer reagent – pH considerations Tris, HEPES, ammonium bicarbonate, PBS, TEAB
Reducing reagents Dithiothreitol (DTT), TCEP (tris(2-carboxyethyl)phosphine)
Depletion of high abundant proteins typically done before digestion Enrichment of post-translational modifications typically done after digestion
Common Extraction Buffers: 4% SDS, 100mM Tris pH 7.6, 100mM DTT 7M urea, 2M thiourea, 0.4M TEAB pH8.5, 20% acetonitrile, 4mM TCEP
freeze + mortar & pestle
Centrifuge insoluble material out & aspirate supernatant (protein extract)
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
Dynamic Range Problem In Samples – A Case for Depletion
Anderson N L , and Anderson N G Mol Cell Proteomics 2002;1:845-867
Dynamic range of proteins in human plasma
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
Reference: http://www.protocol-online.org/forums/index.php?app=core&module=attach§ion=attach&attach_id=889
Beckman Coulter IgY-12 Protein Partitioning Column
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
Reference: Agilent web seminar slides
(MARS)
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
T.Shi et al., Methods (2011), doi:10.1016/j.ymeth.2011.09.001
http://www.sigmaaldrich.com/life-science/proteomics/protein-sample-preparation/protein-depletion-products/seppro-depletion-resins/seppro-igy14-system.html/
Sigma-Aldrich Human IgY14 and SuperMix Columns
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
Enrichment - Post Translational Modifications (PTM’s)
Jensen Nature Reviews Molecular Cell Biology 7, 391-403 (June 2006) | doi:10.1038/nrm1939
Why Enrich for PTM of interest?
In most cases, stoichiometric ratio of PTM species to unmodified species is extremely low.
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
Phosphoproteomic Enrichment
Reference: TE Thingholm, Proteomics 2009, 9, 1451–1468 http://www.ionsource.com/Card/phos/phos.htm
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
Acetylation Enrichment with Anti-Acetyl Lysine Antibody
Reference: KL Guan, Nature Protocols 5, 1583–1595 (2010) doi:10.1038/nprot.2010.117
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
IP Method Considerations
Reference: piercenet.com
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
In-Solution vs. In-Gel Strategies – Dynamic Range In-Solution Prep, 11 proteins In-Gel Prep, 401 proteins
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
† Instrument-specific capabilities required * iTRAQ® Isobaric Tag for Relative and Absolute Quantitation ** SILAC Stable isotope labeling with amino acids in cell culture
INTRO: Protein Quantitation Strategies at the Peptide Level by Mass Spectrometry†
• Discovery-based (complex mixture) – Stable isotope incorporation
• iTRAQ® * • SILAC **
– ‘Label free’ • Spectral counting • Peptide Ion Peak Intensity: XIC-based (extracted ion
chromatogram)
• Targeted analyses – Select peptides of interest – Internal standard for absolute quantitation
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
Isobaric Tag Total mass = 305
Reporter Group 113 –119, 121 m/z
Balance Group (?) Mass 184, 186 – 192 m/z
Amine specific peptide reactive group (NHS) N-hydroxysuccinimide
N
N
O
O
N
O
O
N
N
O
O
N
O
O
~~~~~~~
Applied Biosystems has granted permission to use this slide.
iTRAQ® 8-Plex Reagent Chemical Structure
This will react with primary amines...need to ensure buffer components are compatible or cleaned-up!
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
Isobaric Tags for Relative & Absolute Quantitation (iTRAQ®) Example: Compare Relative Protein Expression Levels in Healthy vs Disease Tissues
Reduce, alkylate Cysteines
Trypsin Digest
Reduce, alkylate Cysteines
Trypsin Digest
Reduce, alkylate Cysteines
Trypsin Digest
Reduce, alkylate Cysteines
Trypsin Digest
Proteolytic Digestion
iTRAQ TAG
114 iTRAQ TAG
115 iTRAQ TAG
116 iTRAQ TAG
117 Label peptides with iTRAQ® Reagents
Tissue Images: Rosas HD et al, (2002) Neurology, 58, 695
MIX
2D LC-MS/MS
Normal Brain Tissue-1
Normal Brain Tissue-2
Obtain protein-containing sample, extract protein
Huntington’s
Disease-1
Huntington’s
Disease-2
Experimental Design for 4-plex iTRAQ® Experiment
50 ug 50 ug 50 ug 50 ug
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
1st Dimension offline HPLC High pH C18 RP
Y. Wang, et al., Proteomics 2011, 11, 2019-2026
2nd Dimension HPLC Inline w/Mass Spec.
Low pH C18 RP
2D Liquid Chromatography-MS/MS
Generated with XCMS by Matt Wroblewski
Try to break down sample complexity
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
SILAC Metabolic Labeling Experimental Workflow
http://www.piercenet.com/method/quantitative-proteomics
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
SILAC Experiment: Proteome Dynamics of B. subtilis in Response to Two Nutritional Challenges
growth on succinate
phosphate starvation
adapted from Soufi B et al; J. Proteome Res. 2010, 9, 3638-3646. Copyright 2010 ACS
Lyse and Combine 1:1
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
© 2015 Regents of the University of Minnesota. All rights reserved.
Label Free and Targeted Quantification Prep
adapted from http://www.piercenet.com/method/quantitative-proteomics
Targeted