QUANTITATIVE LCQUANTITATIVE LC--MS/MS MS/MS Analysis of proteins and peptides Analysis of proteins and peptides

Karolina Krasinska

22

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

3

Absolute quantitation – targeted analysis

Proteolytic digest

(Trypsin, chymotrypsin, AspN etc.)

44

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)

55

Analysis and data processing overview

Q3Collision cellQ1HPLC

Sample Prep

+ digest

X+IS

A) Analysis workflow

B) Data processing

IS

X

66

Triple quadrupole analyzer

MS1 CollisionCell (w/Argon)

MS2

m1

1V 1Vm2

Precursor ion Fragment ion

Scheme from Water Quattro Premiere Training

77

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

88

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?

9

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

10

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

11

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

12

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

13

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

14

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

1515

Assay development outline

• Mass spectrometry (MS)

• Liquid chromatography (LC)

• Sample preparation

• LC-MS/MS method optimization and characterization

1616

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

1717

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

1818

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

1919

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

2020

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

2121

Results – QuanLynx report

IS

Analyte

SRM trace

Calculated concentration

Calibration curve

Analyte and IS area

Signal-to-noise ratio

2222

Most common challenges

• Internal standard (IS) • Matrix effects • Method validation

2323

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

24

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

Res

pons

e

0

50000

100000

150000

200000

250000

300000

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

Res

pons

e

0

500

1000

1500

2000

2500

No IS With IS

25

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

10.0

20.0

30.0

40.0

50.0

60.0

26

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

2727

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

2828

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

29

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

e

0

5000

10000

15000

20000

25000

3030

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

3131

Acknowledgements

Stanford University Mass Spectrometry Staff • Allis Chien• Chris Adams • Pavel Aronov• Theresa McLaughlin

Vincent and Stella Coates Foundation