Quantitative proteomics using TMT® isobaric tags

Dr. Andrew WestonNIHR Biomedical Research Centre for Mental Health

King's College London Proteomics UnitInstitute of Psychiatry

11-13th May 2010

Introduction

• Proteomics: studying the protein complement of an organism or system• Types of experiment:

• protein identification

• profiling

• quantitative screening• single-plex• multiplex:

• isobaric tags (isotopically labelled chemical tags)• isotopic labelling

• targeted quantitative: relative and absolute

• intact protein characterisation

• post-translational modification characterisation

• other e.g. identification of splice variants, stoichiometry of conjugates, multimers

Isobaric tags

• protein chemical labelling technology for quantitation• unlike alternative labelling technologies tags all have the same mass• on fragmentation generate reporter ions of distinct mass• peptides from multiplexed (mixed) samples co-migrate (LC and MS)• quantitative information gained, intensity of reporter ions

e.g. Tandem Mass Tags® (TMT® reagents) from PS Plc:

• Up to 6 variants with incremental isotopic substitutions on reporter region.

Minimum Value (5.0%)

1,174.2?

1,175.5?

1,176.0?1,176.4?

1,177.2?1,177.9?

1,178.4?

1,178.9?

1,179.3?

TMT-127

TMT Parent

y12+2H

y14+2H

y14+2H+1

y14+2H+2

a18-H2O+2H+1

a18-NH3+2H+1

a18-NH3+2H+2

b18-H2O+2H+1

b18-H2O+2H+2

parent+2H-H2O

parent+2H-NH3

y1

b2

y2 y3

b4

y4

b5

y5

b6

y6

b7

y7

y8y9

b9b10

y10

y11

b12y12

b13y13b14

b15

T L S D Y N I Q K+229 E S T L H L V L RR L V L H L T S E K+229 Q I N Y D S L T

m/z

Rel

ativ

e In

tens

ity

0%

25%

50%

75%

100%

0 250 500 750 1000 1250 1500 1750 2000 2250

2,358.39 AMU, +2 H (Parent Error: 32 ppm)

TMT Isobaric tags

���� One spectrum���� Two peptides���� Relative quantity of both

Discovery proteomics: TMT biomarker discovery project

Urine, plasma?

Concentrate?Immunodepletion?

6xplatelet protein extracts (3 of each from subjects before and after aspirin

treatment)

1D gel fractionation (SDS-PAGE)

In-gel digestion and LC-MS/MS

Excision of gel sections

• Label 20µg equivalent with TMT, combine• Add Laemmli buffer for qualitative analysis

Ratio of proteins common to samples

• Differential protein expression in platelets: six-p lex intact protein labelling

Protein profile

Validation of proteomic data

Data analysis

Discovery proteomics

Data analysis - 1

Duplicated experiment

Labelled proteinNon-labelled protein

• 3x3 experiment: platelets from subjects before and after aspirin• Quantities loaded 6x20µg per lane

• Differential protein expression in platelets: six-p lex intact protein labelling

• platelet sample from non-treated individual• Quantity of protein loaded: 20µg

1) Qualitative analysis: how many proteins can be identified?

2) Quantitative analysis

� 404 proteins

Data analysis - 2

Pyruvate kinase isozyme M1/M2Heat shock protein A5 LIMS3 Myosin light polypeptide 6 Clathrin heavy chain 1Actin, cytoplasmic 1 Phosphoglycerate kinase 1 Actin, cytoplasmic 1 Actin, cytoplasmic 1 Tubulin alpha-1B chainTubulin alpha-4A chain Rab GDP dissociation inhibitor alpha Vinculin Rab GDP dissociation inhibitor alpha Heat shock 70 kDa protein 1 Actin, cytoplasmic 1 Isocitrate dehydrogenase [NADP], mitochondrial Thrombospondin-1 Talin-1 Coactosin-like proteinLeukocyte elastase inhibitor Rab GDP dissociation inhibitor alpha Multimerin-1 Rab GDP dissociation inhibitor alpha Rab GDP dissociation inhibitor alpha Glycoprotein lllaMyosin-9 Talin-1 Thrombospondin-1 Tropomyosin alpha-4 chainTalin-1 Coronin-1C_i3 proteinMoesin Myosin-9 Phosphoglycerate kinase 1 Talin-1 Filamin-A Talin-1 14-3-3 protein zeta/delta

Platelet experiment• 195, 255 proteins identified using criteria• Extracted into spreadsheet from identification program, Mascot:

etc

Replicate 2

Replicate 1

� Most peptides detected were quantifiable i.e. TMT-labelled

Data analysis 3: reporter ion intensities

Global analysis of peptide levels:scatterplot showing reporter ion intensities for all spectra assigned to peptides between two conditions

• List of different peptides detected with TMT label attached

• list of spectra collected for each peptide

• Spectrum itself with relative reporter intensity

Suggest down-regulation

Data analysis 3: reporter ion intensities

• Different classes of data: sensitive, resistant; before and after treatment• Complex, global analysis: variation in peptide reporter ion intensity before and after aspirin treatment• OPLS discriminant analysis : variation in response between sensitive and resistant samples after treatment

Multivariate analysis

Contribution to variation between sensitive and res istant

Variation within group

���� Peptides with 10% highest and lowest OPLS weightings taken forward for further statistical analysis.

Increase in resistant compared to sensitive

Decrease in resistant compared to sensitive

• 565 peptides common to both replicates

• Change in peptide expression pre- and post- aspirin treatment in 2x3 subjects (2 sensitive, 4 resistant)

Data analysis 4: quantitative summaries

• Protein level quantitation: boxplots

• Intensity fold change for peptides (≥3) of a protein across 2 experiments i.e. 2 sets of six-plex experiments (each 1 sensitive, 2 resistant aspirin before and after treatment)

Validation of proteomic data

• Western blot• ELISA• Luminex• Selective Reaction Monitoring (SRM or MRM)

Platelet work

� Candidates from TMT-labelling discovery phase chosen for SRM validation

� SRM assays designed for 5 proteins� Simultaneous monitoring of 3 proteins that changed minimally in discovery experiments (“housekeeping”, normalisation)

� Candidates which showed most clear change between subjects examined further by western blotting

MRARPRPRPLWATVLALGALAGVGVGGPNICTTRGVSSCQQCLAVSPMCAW CSDEALPLGSPRCDLKENLLKDNCAPESIEFPVSEAR VLEDRPLSDKGSGDSSQVTQVSPQR IALRLRPDDSKNFSIQVRQVEDYPVDIYYLMDLSYSMKDDLWSIQNLGTKLATQMR KLTSNLR IGFGAFVDKPVSPYMYISPPEALENPCYDMKTTCLPMFGYKHVLTLTDQVT RFNEEVKKQSVSRNRDAPEGGFDAIMQATVCDEKIGWR NDASHLLVFTTDAKTHIALDGRLAGIVQPNDGQCHVGSDNHYSASTTMDYPSLGLMTEKLSQKNINLIFAVTE NVVNLYQNYSELIPGTTVGVLSMDSSNVLQLIVDAYGKIRSKVELEVRDLP EELSLSFNATCLNNEVIPGLKSCMGLKIGDTVSFSIEAKVRGCPQEKEKSFTIKPVGFKDSLIVQVTFDCDCACQAQAEPNSHRCNN GNGTFECGVCRCGPGWLGSQCECSEEDYRPSQQDECSPREGQPVCSQRGECLCGQCVCHSSDFGKITGKYCECDDFSCVRYKGEMCSGHGQCSCGDCLCDSDWTGYYCNCTTRTDTCMSSNGLLCSGRGKCECGSCV CIQPGSYGDTCEKCPTCPDACTFKKECVECKKFDRGALHDENTCNRYCRDE IESVKELKDTGKDAVNCTYKNEDDCVVRFQYYEDSSGKSILYVVEEPECPKGPDILVVLLSVMGAILLIGLAALLIWKLLITIHDR KEFAKFEEERAR AKWDTANNPLYKEATSTFTNITYR GT

1. Selection of peptides to monitor, per protein:

2. Program mass spectrometer to monitor transitions (≥3) (fragmentations) of selected peptides by MS/MS:

3. Inject samples into LC-MS/MS system and monitor relative quantity of protein across samples

Validation of proteomic data – SRM assays

� 4 peptides

� Calculate combined area under curve for transitions, normalise to housekeeping

4. 12 subjects; each peptide monitored, analysis repeated 3x:

5. Boxplot of fold change of peptides per protein between groups

Validation of proteomic data - SRM

Results

• 2 out of 5 proteins reproduced pattern of change seen in discovery phase

• 2 housekeeping proteins remained relatively constant between conditions

• 1 protein taken forward for western blotting

Recent TMT quantitation proteomics publications from CEMS

Other work• comparison of intact and peptide level labelling:Engmann, O., Campbell, J.,Ward, M.,Giese, P.K., Thompson, A.J. (2010) Comparison of a Protein-Level and Peptide-Level Labelling Strategy for Quantitative Proteomics of Synaptosomes Using Isobaric Tags. Journal of Proteome Research.

• comparison of neuronal proteins with/without treatment with potential AD drugsThompson, A. J., Williamson, R., Schofield, E., Stephenson, J., Hanger, D., Anderton, B. (2009). Quantitation of glycogen synthase kinase-3 sensitive proteins in neuronal membrane rafts. PROTEOMICS 9(11); 3022-3035.

• in press: identification of candidate plasma-based biomarker in discovery proteomics experiment; set of plasma samples taken from AD patients at different rates of decline.� TMT labelling of plasma proteins in 45 subjects � 152 proteins, 52 with quantitative data, 2 showing greatest difference (non-disease vs. disease) validated with western blotting.

Platelet biomarker work• commercial application note : Discovery and evaluation of candidate markers of aspirin resistance using intact TMT labelling

• paper : discovery and validation of candidate biomarkers using discovery proteomics, SRM mass spec., western blotting

• grant application : pilot data used for in collaboration with Cardiovascular Division at King’s

Acknowledgements

Tim Goodman, Dr. Emma Schofield – discovery proteomics

Dr. Silke Becker, Dr. Emma Schofield, Prof. Albert Ferro – SRM validation work

Dr. Malcolm Ward – oversight of both proteomics projects

Proteome Sciences Plc – TMT reagents, personnel

NIHR Biomedical Research Centre – mass spectrometry equipment for SRM, funding

• Email:• Andrew Weston: andrew.weston@kcl.ac.uk

• Steven Lynham: steven.lynham@kcl.ac.uk

• Malcolm Ward: malcolm.ward@kcl.ac.uk

• KCL Centre of Excellence for Mass Spectrometry (CEMS) website: http://www.kcl.ac.uk/research/facilities/mspec/

• Drop-in clinic:• IOP events: http://www.iop.kcl.ac.uk/events/• notification list: proteomics_kcl-subscribe@yahoogroups.com

• Telephone: 020 7848 0248

Further information