QUANTITATIVE LC-MS/MS Analysis of proteins and peptides · 2014-02-11 · • Quantitation on...
Transcript of QUANTITATIVE LC-MS/MS Analysis of proteins and peptides · 2014-02-11 · • Quantitation on...
QUANTITATIVE LCQUANTITATIVE LC--MS/MS MS/MS Analysis of proteins and peptides Analysis of proteins and peptides
Karolina Krasinska
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Overview• Introduction: Quantitative approach to protein
and peptide analysis • Background: Instrumentation, workflow• Assay development: Step by step • Quantitative LC-MS/MS assay: method,
results • Common issues:
– Matrix effect – Internal standard (IS) – Method validation – Analyte stability
• Quantitation on peptide level
• Know what you are going to quantify: – well characterized protein – protein ID via LC-IT-MS/MS – in silico digest based on theoretical AA sequence
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Absolute quantitation – targeted analysis
Proteolytic digest
(Trypsin, chymotrypsin, AspN etc.)
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Sample• Protein – unlimited size (gel, solution,
biological matrix) • Peptides – ideally 6-12 aa (up to 30-40 aa);
may be phosphorylated • Biological matrices:
– plasma, serum, erythrocytes– cerebrospinal fluid (CSF)– urine – bile – cell culture media – plant and animal tissues (e.g. leaf, brain, liver)
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Analysis and data processing overview
Q3Collision cellQ1HPLC
Sample Prep
+ digest
X+IS
A) Analysis workflow
B) Data processing
IS
X
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Triple quadrupole analyzer
MS1 CollisionCell (w/Argon)
MS2
m1
1V 1Vm2
Precursor ion Fragment ion
Scheme from Water Quattro Premiere Training
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Advantages of SRM scanning mode
• MS/MS provides higher sensitivity and selectivity, enabling
– Less extensive sample preparation– Greater sensitivity via increased selectivity – Use of shorter HPLC columns – Use of shorter run times and higher sample
throughput
• Translates into time and effort savings, plus a more sensitive method
4.00 5.00 6.00 7.00Time0
100
%
5.61
m/z100 150 200 250 300 350 400 450 500
%
0
100 178.0
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Before we start – checklistQuestions:
• What is the protein of interest? (MW, sequence, PTMs) • Number of peptides included in the assay? • Sample types? (matrices) • Desired/required LLOQ and calibration range? • Standards availability? (purified protein, peptides and labeled peptides) • Purpose of developing the assay? (preliminary studies, confirmation of findings from different methods, publication) • Available funding and timelines?
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Peptide selection rules
Main rules: • 6-12 AA optimal (up to 30-40aa) • No chemically reactive residues (Trp, Met, Cys) • No ragged ends (2xR; RK; 2xK) • No potential PTM• unique sequenceAlso consider:• Preferably containing P (dominant cleavages)• If MS/MS data already available than look for peptides giving high
intensity fragment ions of m/z higher than precursor ion (noise reduction)
• R in P proximity (potential missed tryptic cleavage)• Select multiple peptides for each protein whenever possible
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Protein X - fragmentDigest Table
# Pos Mass pI Peptide
1 1213.50 9.50 MAAVAALQLGLR
2 543.62 9.79 AAGLGR
3 828.93 9.79 APASAAWR
4 473.57 9.47 SVLR
5 1464.69 12.30 VSPRPGVAWRPSR
6 1082.15 5.99 CGSSAAEASATK
7 3203.69 4.03 AEDDSFLQWVLLLIPVTAFGLGTWQVQR
8 174.20 9.75 R
9 146.19 8.75 K
10 332.40 8.75 WK
11 1157.33 4.53 LNLIAELESR
12 1719.07 4.14 VLAEPVPLPADPMELK
13 1018.18 8.59 NLEYRPVK
14 273.34 9.72 VR
15 792.86 6.73 GCFDHSK
16 939.16 6.10 ELYMMPR
Digest Table
# Pos Mass pI Peptide
17 816.97 5.50 TMVDPVR
18 374.40 6.10 EAR
19 2654.93 5.32 EGGLISSSTQSGAYVVTPFHCTDLGK
20 146.19 8.75 K
21 714.78 5.97 VNPETR
22 274.32 8.75 QK
23 1515.74 4.14 GQIEGEVDLIGMVR
24 618.69 6.00 LTETR
25 1326.43 4.53 QPFVPENNPER
26 911.98 8.76 NHWHYR
27 804.92 4.37 DLEAMAR
28 2744.06 4.37 ITGAEPIFIDANFQSTVPGGPIGGQTR
29 487.60 9.72 VTLR
30 2791.16 6.75 NEHLQYIVTWYGLSAATSYLWFK
31 146.19 8.75 K
32 434.54 9.75 FLR
33 429.47 5.52 GTPGV
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Digest Table
# Pos Mass pI Peptide
1 1213.50 9.50 MAAVAALQLGLR
2 543.62 9.79 AAGLGR
3 828.93 9.79 APASAAWR
4 473.57 9.47 SVLR
5 1464.69 12.30 VSPRPGVAWRPSR
6 1082.15 5.99 CGSSAAEASATK
7 3203.69 4.03 AEDDSFLQWVLLLIPVTAFGLGTWQVQR
8 174.20 9.75 R
9 146.19 8.75 K
10 332.40 8.75 WK
11 1157.33 4.53 LNLIAELESR
12 1719.07 4.14 VLAEPVPLPADPMELK
13 1018.18 8.59 NLEYRPVK
14 273.34 9.72 VR
15 792.86 6.73 GCFDHSK
16 939.16 6.10 ELYMMPR
Digest Table
# Pos Mass pI Peptide
17 816.97 5.50 TMVDPVR
18 374.40 6.10 EAR
19 2654.93 5.32 EGGLISSSTQSGAYVVTPFHCTDLGK
20 146.19 8.75 K
21 714.78 5.97 VNPETR
22 274.32 8.75 QK
23 1515.74 4.14 GQIEGEVDLIGMVR
24 618.69 6.00 LTETR
25 1326.43 4.53 QPFVPENNPER
26 911.98 8.76 NHWHYR
27 804.92 4.37 DLEAMAR
28 2744.06 4.37 ITGAEPIFIDANFQSTVPGGPIGGQTR
29 487.60 9.72 VTLR
30 2791.16 6.75 NEHLQYIVTWYGLSAATSYLWFK
31 146.19 8.75 K
32 434.54 9.75 FLR
33 429.47 5.52 GTPGV
Protein X - fragment
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Digest Table
# Pos Mass pI Peptide
1 1213.50 9.50 MAAVAALQLGLR
2 543.62 9.79 AAGLGR
3 828.93 9.79 APASAAWR
4 473.57 9.47 SVLR
5 1464.69 12.30 VSPRPGVAWRPSR
6 1082.15 5.99 CGSSAAEASATK
7 3203.69 4.03 AEDDSFLQWVLLLIPVTAFGLGTWQVQR
8 174.20 9.75 R
9 146.19 8.75 K
10 332.40 8.75 WK
11 1157.33 4.53 LNLIAELESR
12 1719.07 4.14 VLAEPVPLPADPMELK
13 1018.18 8.59 NLEYRPVK
14 273.34 9.72 VR
15 792.86 6.73 GCFDHSK
16 939.16 6.10 ELYMMPR
Digest Table
# Pos Mass pI Peptide
17 816.97 5.50 TMVDPVR
18 374.40 6.10 EAR
19 2654.93 5.32 EGGLISSSTQSGAYVVTPFHCTDLGK
20 146.19 8.75 K
21 714.78 5.97 VNPETR
22 274.32 8.75 QK
23 1515.74 4.14 GQIEGEVDLIGMVR
24 618.69 6.00 LTETR
25 1326.43 4.53 QPFVPENNPER
26 911.98 8.76 NHWHYR
27 804.92 4.37 DLEAMAR
28 2744.06 4.37 ITGAEPIFIDANFQSTVPGGPIGGQTR
29 487.60 9.72 VTLR
30 2791.16 6.75 NEHLQYIVTWYGLSAATSYLWFK
31 146.19 8.75 K
32 434.54 9.75 FLR
33 429.47 5.52 GTPGV
Protein X - fragment
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Digest Table
# Pos Mass pI Peptide
1 1213.50 9.50 MAAVAALQLGLR
2 543.62 9.79 AAGLGR
3 828.93 9.79 APASAAWR
4 473.57 9.47 SVLR
5 1464.69 12.30 VSPRPGVAWRPSR
6 1082.15 5.99 CGSSAAEASATK
7 3203.69 4.03 AEDDSFLQWVLLLIPVTAFGLGTWQVQR
8 174.20 9.75 R
9 146.19 8.75 K
10 332.40 8.75 WK
11 1157.33 4.53 LNLIAELESR
12 1719.07 4.14 VLAEPVPLPADPMELK
13 1018.18 8.59 NLEYRPVK
14 273.34 9.72 VR
15 792.86 6.73 GCFDHSK
16 939.16 6.10 ELYMMPR
Digest Table
# Pos Mass pI Peptide
17 816.97 5.50 TMVDPVR
18 374.40 6.10 EAR
19 2654.93 5.32 EGGLISSSTQSGAYVVTPFHCTDLGK
20 146.19 8.75 K
21 714.78 5.97 VNPETR
22 274.32 8.75 QK
23 1515.74 4.14 GQIEGEVDLIGMVR
24 618.69 6.00 LTETR
25 1326.43 4.53 QPFVPENNPER
26 911.98 8.76 NHWHYR
27 804.92 4.37 DLEAMAR
28 2744.06 4.37 ITGAEPIFIDANFQSTVPGGPIGGQTR
29 487.60 9.72 VTLR
30 2791.16 6.75 NEHLQYIVTWYGLSAATSYLWFK
31 146.19 8.75 K
32 434.54 9.75 FLR
33 429.47 5.52 GTPGV
Protein X - fragment
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Peptide selection – “good spectrum”
447.6?
y8-H2O+2H
y8+2H
y7+1parent+2H-H2O
y1
b2y2
b3
y3
y4
b4
y5
b5
y6b6
b7
y7
b8
y8
b9 y9
L L Q F V T G T S RR S T G T V F Q L L
m/z
Rel
ativ
e In
tens
ity
0%
25%
50%
75%
100%
0 200 400 600 800 1000
1,120.90 AMU, +2 H (Parent Error: 250 ppm)
y6+1
b3y3 b4y4 b5y5
y6
b6
y7
b7
y8
b9y9 b10
y10b11 b12
E G F F E L I P Q D L I KK I L D Q P I L E F F G E
m/z
Rel
ativ
e In
tens
ity
0%
25%
50%
75%
100%
0 250 500 750 1000 1250 1500
1,548.30 AMU, +2 H (Parent Error: 310 ppm)
• Excellent sequence coverage• High dispersion of energy • Good for peptide ID
• Good sequence coverage• Low dispersion of energy • Good for quantitation
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Assay development outline
• Mass spectrometry (MS)
• Liquid chromatography (LC)
• Sample preparation
• LC-MS/MS method optimization and characterization
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Method development - mass spectrometry
• Acquisition of MS and MS/MS spectra for standard solution of the peptide
– 10-50µM, – direct infusion
• Precursor ion – fragment ion MS parameters optimization
– most efficient ionization; precursor ion preferably +2– most efficient fragmentation; fragment ions preferably
with m/z higher than precursor ion• Method set up and test run
– Choose three precursor ion – fragment ion pairs for each peptide
MS
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Liquid chromatography
• HPLC column– RP type column – C18– Retention – Separation – usually no need to have a base
peak separation – Gradient elution – MS compatible solvents and buffer modifiers
(0.1% FA in water, 0.1% FA in acetonitrile)– Shortest runs possible (usually 4-8 min per
injection)
LC
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LC-MS method optimization & characterization
• Linear calibration curve • Limit of detection (LOD)
– 3:1 signal-to-noise ratio• Limit of quantitation (LOQ)
– 10:1 signal-to-noise ratio• Sample matrix interferences
– analysis of blank sample matrix spiked with pure standard
• Carryover • Stability of the analyte
LC-MS
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Sample preparation• Objectives:
– Isolating analyte from matrix – Removing contaminants, desalting – Concentrating analyte if necessary – Reconstituting in appropriate LC-MS compatible reagent
• Extraction methods: – Prior to proteolytic digest
• Protein precipitation– Methanol– Acetonitrile
• Immunoprecipitation– After proteolytic digest
• Solid Phase Extraction (SPE)– C18 – Ion exchange (e.g. SCX)
– Combination of the above
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Quantitative LC-MS/MS assayEach set of analyzed samples contains the following: • 6-8 point calibration curve sets (in triplicate).
A calibration curve set is usually run at the beginning, at the end, and once or more during the sample set.
• 1 QC per 10 samples (in triplicate) • n# of samples (in triplicate) • blank injections if necessary
Q3Collision cellQ1HPLC
IS
X
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Results – QuanLynx report
IS
Analyte
SRM trace
Calculated concentration
Calibration curve
Analyte and IS area
Signal-to-noise ratio
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Most common challenges
• Internal standard (IS) • Matrix effects • Method validation
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Internal standard – to use or not to use?
• Benefit of IS:– If the analyte and IS suffer the same losses and the same
effects in the matrix, matrix effects and sample losses cancel when we take the ratio of IS to analyte
• IS compensates for common analyte losses: – Analyte adsorption on surfaces– Extensive sample manipulation – Degradation – Evaporation – Autosampler variability IS
X
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Peptide adsorption on the surface
Compound name: PSMB2Correlation coefficient: r = 0.973449, r^2 = 0.947603Calibration curve: 746.428 * x + -726.173Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
fmol/ul0 50 100 150 200 250 300 350 400
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pons
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Compound name: PSMB2Correlation coefficient: r = 0.994609, r^2 = 0.989247Calibration curve: 6.64019 * x + -1.62103Response type: Internal Std ( Ref 10 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
fmol/ul0 50 100 150 200 250 300 350 400
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pons
e
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No IS With IS
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Extreme exampleCompound name: OligoACorrelation coefficient: r = 0.852161, r^2 = 0.726178Calibration curve: 20.9105 * x + -1.15707Response type: Internal Std ( Ref 2 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
pmol/ul0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00
Res
pons
e
0.0
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60.0
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Tricks to improve calibration curve linearity
• Isotopically labeled internal standard• Sample buffer modification:
– Sample matrix – Diluted sample matrix – Addition of neutral protein – Higher organic solvent content
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Matrix effects• Matrix effects:
– Ion suppression – Interferences from metabolites – Signal enhancement
• Assessment of matrix effect: – Post-column infusion of analyte – Comparison of analyte in matrix-free solution vs. spiked
blank matrix – extraction efficiency assessment • Minimizing matrix effects:
– Use isotopically labeled internal standard – Use fragment ions of m/z higher than precursor ion– Generate “cleaner” extract – Optimize HPLC method
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Method Validation• Detection capability:
– LOD – signal to noise ratio 3:1 – LOQ – signal to noise ratio 10:1
• Calibration curve – linear dynamic range • Precision and accuracy • Selectivity • Specificity • Stability of analyte (and matrix):
– Short-term, long-term – Low and high concentrations– Analyte in sample solvent and in raw matrix – Dry extract, reconstituted standard/extract– Freeze/thaw cycles
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Analyte stability
No ISWith ISCompound name: ProgCorrelation coefficient: r = 0.999252, r^2 = 0.998505Calibration curve: 0.440538 * x + 0.0156705Response type: Internal Std ( Ref 2 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
fmol/ul0.00 1.00 2.00 3.00 4.00 5.00
Res
pons
e
0.00
0.50
1.00
1.50
2.00
Compound name: ProgCorrelation coefficient: r = 0.970733, r^2 = 0.942323Calibration curve: 4511.2 * x + 221.327Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
fmol/ul0.00 1.00 2.00 3.00 4.00 5.00
Res
pons
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Literature References
1. Mass Spectrometry: Principles and Applications by Edmond de Hoffmann, Vincent Stroobant
2. Trace Quantitative Analysis by Mass Spectrometryby Robert K. Boyd, Cecilia Basic, Robert A. Bethem
3. LC/MS: A practical User’s Guide by Marvin McMaster 4. Quantitative Proteomics by Mass Spectrometry by
Salavatore Sechi5. www.ionsource.com6. SUMS website: mass-spec.stanford.edu
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Acknowledgements
Stanford University Mass Spectrometry Staff • Allis Chien• Chris Adams • Pavel Aronov• Theresa McLaughlin
Vincent and Stella Coates Foundation