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Introduction to Mass spectrometry Based Proteomics Workflow
What is proteomics?
Proteomics includes not only the identification and quantification of proteins, but also the determination of their localization, modifications, interactions, activities, and, ultimately, their function.
-Stan Fields in Science, 2001.
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Dynamic Range =103
How to think about mass spectrometry based proteomics
mRNAAble to amplify (PCR)
Protein No amplification
High variability in amount (>109)
How to think about mass spectrometry based proteomics
What it is: A highly powerful tool for protein identification and quantification Complementary to other technologies and analysis methods What it is not: Magic Able to give all the answers Simple (relatively speaking) Cheap
Mass Spectrometry based proteomics: What it is and what it isnt
What can mass spectrometry tell me?
proteins in mixtures quantitative analysis of protein expression post-translational modifications:
Phosphorylation
protein interactions
Mass Spectrometry PrimerA mass spectrometer
measures mass to charge ratio or m/z
Ionization Mass Separation Ion collection
quadrupole ion trap time-of-flight
MALDI Electrospray mass analysis
Protein, MW = 10,000 +
digest into peptides cleaves C-teminal side of arginine (R) and lysine (K)
TrypsinN-THK.NCPHIVVGTPGR.IPD-C
What do we put in our mass spectrometer to measure m/z?
NH2NH2
NH2
-COOHR
K-COOH
-COOHK
Peptides, MW < 4,000
+
+
+
+
+
+
High voltage placed on a fused silica column causes a spray of charged droplets which evaporate leaving charged peptides
NewObjective
UniversityofBristol
NH2 K-COOH++
Electrospray ionization (ESI)
Matrix-assisted laser desorption/ionization (MALDI)
3 mm
matrix sampleCOOH
OH
CH CNC
UV laser
A laser pulse excites the matrix material at its resonant frequency and energy is imparted to peptides, charging them.
Triple Quadrupole Mass SpectrometerTriple Quadrupole Mass Spectrometer
electrospray sourceelectrospray sourceIonizationIonization
Q1Q1 Q2Q2 Q3Q3
collision cellcollision cellMass SelectionMass Selection
DetectorDetectorIon CollectionIon Collection
microcapillarymicrocapillary
Quadrupole Optics
In a quadrupole mass spectrometer four (quad) parallel rods (poles) are arranged equidistantly from a central (imaginary) axis.
Charged ions are injected along the central axis of the quadrupole assembly.
Static and alternating (radio frequency) electric potentials are applied to opposite pairs of rods, creating a fluctuating electric field.
m/z
NN
N
N
N
Mass Spectrometers identify peptides by fragmenting along the amide backbone
++NSGDIVNLGSIAGRNSGDIVNLGSIAGR++
b yb y
++N SGDIVNLGSIAGRN SGDIVNLGSIAGR++++NS GDIVNLGSIAGRNS GDIVNLGSIAGR++++NSG DIVNLGSIAGRNSG DIVNLGSIAGR++++NSGD IVNLGSIAGRNSGD IVNLGSIAGR++++NSGDIV NLGSIAGRNSGDIV NLGSIAGR++++NSGDIVN LGSIAGRNSGDIVN LGSIAGR++++NSGDIVNL GSIAGRNSGDIVNL GSIAGR++++NSGDIVNLG SIAGRNSGDIVNLG SIAGR++++NSGDIVNLGS IAGRNSGDIVNLGS IAGR++++NSGDIVNLGSIA GRNSGDIVNLGSIA GR++++NSGDIVNLGSIAG RNSGDIVNLGSIAG R++
Fragmentation occurs along the backbone, revisited
Y ions
++NSGDIV NLGSIAGRNLGSIAGR++
RRGGIISS AAGGLLNN
++NN+ + NSNS+ + NSGNSG+ + NSGDNSGD+ + NSGDI NSGDI + + NSGDIVNSGDIV+ + NSGDIVNNSGDIVN+ + NSGDIVNLNSGDIVNL+ + NSGDIVNLGNSGDIVNLG+ + NSGDIVNLGSNSGDIVNLGS+ + NSGDIVNLGSINSGDIVNLGSI+ + NSGDIVNLGSIANSGDIVNLGSIA+ + NSGDIVNLGSIAGNSGDIVNLGSIAG+ + RR+ + RGRG+ + RGARGA+ + RGAIRGAI+ + RGAISRGAIS+ + RGAISGRGAISG+ + RGAISGLRGAISGL+ + RGAISGLN RGAISGLN + + RGAISGLNVRGAISGLNV+ + RGAISGLNVIRGAISGLNVI+ + RGAISGLNVIDRGAISGLNVID+ + RGAISGLNVIDGRGAISGLNVIDG+ + RGAISGLNVIDGSRGAISGLNVIDGS
++NSGDIVNLGSIAGRNSGDIVNLGSIAGR++
bions
bions
yions
yions
The fragment ladder allows sequence assignment
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m/zm/z
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HH22 NN --NSGDIVNLGSIAGRNSGDIVNLGSIAGR--COOHCOOH
GGDD
II
VVNN
LLGGSS
IIAAGGRR
NN SSGG
DDII
VV NN LLGG
Quadrupole Optics cont.
The quadrupole can function in a second mode called tandem The quadrupole can function in a second mode called tandem mass spectrometry or MS/MS.mass spectrometry or MS/MS.
A particular peak is chosen from a MS scan and the first quad A particular peak is chosen from a MS scan and the first quad allows only that m/z to pass into the second quad.allows only that m/z to pass into the second quad.
The second quad accelerates the species through a voltage The second quad accelerates the species through a voltage causing collisions with an inert gas present.causing collisions with an inert gas present.
If the ion is a peptide, the collisions cause bond breakage If the ion is a peptide, the collisions cause bond breakage selectively at the selectively at the amideamide bondsbonds
The charged fragments enter the third quad which performs a MS The charged fragments enter the third quad which performs a MS scan generating a unique pattern associated with the fragments scan generating a unique pattern associated with the fragments and thus the parent peptideand thus the parent peptide
These fragments can be deconvoluted to give a peptide sequenceThese fragments can be deconvoluted to give a peptide sequence
Ar
Ar600600 800800 10001000 12001200 14001400
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peptides
trypsin
gel
Tandem mass spectrometry
ESIESI
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tandem mass spectrumtandem mass spectrum
fragment peptide
Ar
Ar
ArAr
1. MS survey scan
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Sample PeptideSample Peptide
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HH22 NN --NSGDIVNLGSIAGRNSGDIVNLGSIAGR--COOHCOOH
GGDD
II
VVNN
LLGGSS
IIAAGGRR
NN SSGG
DDII
VV NN LLGG
Making an identification by database searching using SEQUEST
SEQUEST is a search program that assigns a peptide sequence to a spectra by comparing it to virtual spectra from a protein database
1.1. An MS/MS scan of m/z 750 and charge 2+An MS/MS scan of m/z 750 and charge 2+ the the molecular weight is 1500 Damolecular weight is 1500 Da
2.2. SEQUEST searches a protein database starting at the SEQUEST searches a protein database starting at the first amino acid to find all possible peptides that weight first amino acid to find all possible peptides that weight 1500 +/1500 +/-- 1.51.5
3.3. SEQUEST fragments each virtually and compares to the SEQUEST fragments each virtually and compares to the experimental spectra. experimental spectra.
4.4. For a good spectra, one peptide stands out from all For a good spectra, one peptide stands out from all othersothers
SEQUEST ExampleSEQUEST Example
Scoring a match
4 5 6 7 83210 9 10 11
4 5 6 7 83210 9 10 11
4 5 6 7 83210 9 10 11
4 5 6 7 83210 9 10 11
4 5 6 7 83210 9 10 11
4 5 6 7 83210 9 10 11
NSGDIVNLGSIAGR NSADIVNLGSIAGR
Acquired spectra
Theoretical spectra
Dotproduct
The linear ion trap LTQ
The Orbitrap and LTQ-FT
Average vs. monoisotopic mass
LTQ-FT
Time of Flight
All ions are imparted with the same energy (E) by the application of an electric field.
E=mv2/2
m=2E/v2
Distance/time=v
m=2E/(d/t)2
Mass can be calculated based on time of arrival because the energy, flight tube length, and time are
known.
Scanning with a ESI-tof
High mass accuracy Low duty cycle Large data files
ESIESI
Electron Transfer Dissociation
PNAS February 13, 2007 vol. 104 no. 7 21932198
C
ETD
Source: Thermo Fisher
ETD
ETD
Quantitative Proteomics
Why?...Comparisons
Particularly well suited to answering biologic questions
Detail protein level changes to a biologic stimulus Used as a tool to evaluate protein distributions Used as a tool to follow complex formation
Modified from CEBI web site
Isotopic modification strategies
Mass spectrometers measure mass-hence isotopically different peptides can be compared.
Single ion chromatograms for each isotope can be compared to determine relative quantities of each.
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lightlight heavyheavy
The general formats of isotope addition
Metabolic labeling Tagging of Cysteine Tagging of free amines 18O labeling during digestion Spiking in a heavy peptide to quantify a target
Isotopic tags
Label should react with a group common in proteins
Labeling must be complete Labeling must be stable Label cannot inhibit ionization or fragmentation Label must provide a large enough mass
difference for peaks to be resolved. Light and heavy peptides should co-elute in all
separation steps preceding mass spectrometry
Stable Isotope Labeling with Amino acids in Cell culture
SILAC
Cells are grown in media lacking Arginine or Lysine
These amino acids are supplied as either heavy (13C6 ,
15N4 Arginine and 13C6 Lysine) or light into media Serum is dialyzed to remove amino
acids
SILAC
Heavy Lysine,
Arginine or both
Unmodified amino acids
+
Media w/o Lys and Arg
FCS dialyzed to remove amino acids
Stimulus of
interest
Control
Lyse cells together
SILAC
Very simple, grow cells and lyse together. Dialyzed serum may cause growth issues in some
cell lines All peptides are labeled- this can challenge the
mass spec duty cycle in some experiments Not applicable to cells that cannot be easily
propagated i.e. primary cells Not applicable to samples where propagation is
not possible i.e. mammalian serum or organs
Protein Labeling
Isotopic tagging at the protein level is most commonly done through cysteine or lysine modification.
A variety of reactants are available that incorporate thiol specific chemistry with variable isotopic tags. Some are selective
Amine based reagents are available as well.
Cleavable Isotope Coded Affinity Tags
Heavy reagent: 13CHeavy reagent: 13C--ICAT ICAT Light Light reagentreagent: 12C: 12C--ICATICAT
S
N N
O
SH
cys?I
NO
ON
O
NPeptide
O
ON
OS
H2 N
Acid cleavage
Protein Quantification and Identification by the ICAT StrategyProtein Quantification and Identification by the ICAT Strategy
Affinity Affinity separation separation with Avidinwith Avidin
Combine and Combine and proteolyzeproteolyze
ICATICAT-- labeled labeled
cysteinescysteines
Mixture 2Mixture 2
Mixture 1Mixture 1
ProteinsProteinsPeptidesPeptides
ICAT
Selective and therefore reduces complexity potentially increasing dynamic range
Most proteins have a cysteineHowever:
Method involves a variety of handling steps that can cause sample loss
Parallel cell lysis and handling can introduce bias.
Peptide level labeling
amine based prototypes N-isotag iTRAQ
Can be amine or cysteine based
iTRAQ
iTRAQ (isotope Tags for Relative and Absolute Quantitation).
Commercially available from ABI Requires CID to generate reporter ions for
quantitation. Requires a TOF instrument
NH2
NH2
NH2
NH2
iTRAQ schema
CID of iTRAQ labeled peptides
Copyright 2004 American Society for Biochemistry and Molecular Biology
Ross, P. L. (2004) Mol. Cell. Proteomics 3: 1154-1169
ITRAQ
Copyright 2004 American Society for Biochemistry and Molecular Biology
Ross, P. L. (2004) Mol. Cell. Proteomics 3: 1154-1169
ITRAQ
Copyright 2004 American Society for Biochemistry and Molecular Biology
Ross, P. L. (2004) Mol. Cell. Proteomics 3: 1154-1169
Labelling is consistent within one protein
iTRAQ summary
Very effective method for multiplexed quantitative studies
Trades off increased ID/quantification using many peptides vs. selection strategy (ICAT)
Is not compatible with LC/MS strategies that choose CID peaks based on pair intensity ratios
Label Free Quantitative Proteomics
Isotopes are not used Quantification is performed computationally by comparing
sequential runs Metrics of comparison are typically AUC for aligned runs MS/MS is either incorporated or done later as targeted
MS/MS As an alternative, peptide counting can be used as a method
for relative quantification with MS/MS experiments.
How is this technology implemented?
Large scale
Small scale
Medium scale
Big project example
4000 proteins
>300 orbitrap runs
Big group
Big $$$$$$
Medium Scale Proteomics
The key to medium scale proteomics is a mechanical or affinity based preparation that reduces the complexity of the starting material.
Examples: centrifugation, precipitation.
Peroxisome membrane
We have combined classical subcellular fractionation with large-scale quantitative mass spectrometry to identify proteins that enrich specifically with peroxisomes of Saccharomyces cerevisiae.
Micro-Proteomics
Focus on a particular protein complex Affinity based purification Takes advantage of existing technology Results more approachable for an individual
investigator
Micro-Proteomics
Yknown
unknown
Release
Identification of binding partners
Conclusions 1
Proteomics is rapidly advancing: Relative quantification is here. Large scale experiments are becoming easier with
better automation tools, BUT they generate vast amounts of data and consume significant resources.
Medium and small scale projects can be approached by an individual investigator here and now.
Phosphorylation is observable but methodology is still under development.
Conclusions 2 Mass spectrometers are fantastic
The results you get out are determined by what you put in.
The results you get out are determined by what you put in
The results you get out are determined by what you put in.
Problems are most often NOT the result of poor instrument performance
Your results depend on the following: The purity and cleanliness of you preparation The complexity of your sample compared to the dynamic range and duty cycle of the instrument
Introduction to Mass spectrometry Based Proteomics Workflow What is proteomics?How to think about mass spectrometry based proteomicsHow to think about mass spectrometry based proteomicsMass Spectrometry based proteomics: What it is and what it isntWhat can mass spectrometry tell me?Mass Spectrometry PrimerWhat do we put in our mass spectrometer to measure m/z?Electrospray ionization (ESI)Slide Number 10Slide Number 11Quadrupole OpticsSlide Number 13Slide Number 14Mass Spectrometers identify peptides by fragmenting along the amide backbone Fragmentation occurs along the backbone, revisitedY ionsSlide Number 18The fragment ladder allows sequence assignmentQuadrupole Optics cont.Slide Number 21Slide Number 22Making an identification by database searching using SEQUESTSlide Number 24Scoring a matchThe linear ion trap LTQThe Orbitrap and LTQ-FTSlide Number 28Average vs. monoisotopic massLTQ-FTSlide Number 31Time of FlightSlide Number 33Scanning with a ESI-tofSlide Number 35Electron TransferDissociationETDETDETDQuantitative ProteomicsWhy?...ComparisonsSlide Number 42Isotopic modification strategiesThe general formats of isotope additionIsotopic tagsStable Isotope Labeling with Amino acids in Cell cultureSILACSILACSILACProtein LabelingCleavable Isotope Coded Affinity TagsSlide Number 51ICATPeptide level labelingiTRAQiTRAQ schemaCID of iTRAQ labeled peptidesSlide Number 57Slide Number 58Slide Number 59iTRAQ summaryLabel Free Quantitative Proteomics How is this technology implemented?Slide Number 63Big project exampleMedium Scale ProteomicsPeroxisome membraneMicro-ProteomicsMicro-ProteomicsConclusions 1Conclusions 2
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