Protein & PTM Profiling and Identification Core

Yale/NIDA Neuroproteomics Center EAB Meeting

TuKiet T. Lam, PhD

May 1st, 2019

Mission and Operating Principle

Center Investigators

Instrumentation

Knowledge &Expertise

TechnologyDevelopment & Implementation

Support

Cost recoveryCenter Investigators Projects (90% Center funds, 10% Investigator funds)

Pilot Projects (100% Center funds)

Thermo Fisher Scientific nano-UPLC ESI LTQ-Orbitrap ELITE MS systems

Agilent 1200 UPLCAB Sciex 4000 QTRAP

MS system

Mass Spectrometers currently located within the Core

ELITE

Thermo Fisher Scientific nano-UPLC ESI Q-Exactive Plus MS systems

Thermo Fisher Scientific nano-UPLC ESI Orbitrap Fusion MS systems

Thermo Fisher Scientific nano-UPLC ESI LTQ-Velos MS systems

For Protein ID For Protein PTM, Profiling, & Quantitation

For Metabolism Separation & Quantitation

For Open Access Usage

Waters UPLC (H-Class)

Q-Exactive HF-X mass spectrometer with ACQUITY UPLC M-Class.

New Instrumentation

Instrumentation

ResolutionMass Accuracy

Fragmentation Capabilities

Mass Spectrometer Capabilities

H2N C C NOR1

C C NORn-1

C CORn

OH

m+nHn+

y1

bn-1

z1·

cn-1

...

ETD

CID/HCDRetention of labile modificationsNo X-P cleavage

Facile loss of H3PO4X-P cleavage preferred

m/z 434432430428

429.22623

Deuterated (D)

Protonated (P)

430.22990431.23346

429.22657

430.22835

430.23262

431.23617 432.23963P

DD

D

P

P

P

P

220 260 300 340 380 m/z

263 264 265 266 267m/z

265.04689 (Exp.)

Zoom

265.04713 (Cal.)

0.00024 (Diff.)- 0.9 ppm (Error)

Quantitation

40 80 120 160 200 240 280 320 360 400m/z, Da

62.2

326.8

309.7

265.3

1.2 1.6 2.0 2.4 2.8 3.2 3.6 4.0Time, min

2.03 min

Insturment QE-Focus QE QE-Plus QE-HF QE-HFXQE-

UHMR O-FusionO-Fusion

Lumos O-ID-XMax resolution (FWHM)

@m/z 20070k 140k 140k

(Opt. 280k)240k 240

200k at m/z 400

500k 1,000k 7.5-500k

Mass accuracy, (internal)Mass accuracy (external)

Mass range50 to 3,000 m/z

350 to 80,000

m/z50-2,000 m/z

Dynamic Range - - -

Scan ratesUp to 18

HzUp to 40

HzUp to 12

Hz20Hz 40Hz

30Hz Orbitrap 40Hz ion trap

Polarity switch <1.1sec <1.1sec 1.1 SecPRM -

Multiplex (precursor/ scan) - 10 20 10Decision-tree (CID/HCD/

ETD)ETD option Yes

Yes

<1 ppm<3 ppm

Yes<1 sec

10

50 to 6,000 m/z 50 to 8,000 m/z

>5000:1

Up to 12 Hz

Comparison of Thermo MS (QE and Orbitrap)

From Thermo Fisher Scientific Brochure

Mass Spectrometry and Proteomics Analyses

Protein ID(Gel & Solution)

Protein PTM(e.g. Phosphorylation

and acetylation)

Intact Protein Mass*

HRMS(Exact Mass)

Label Free Quantitation (DDA & DIA)

iTRAQ& TMT SILAC

Parallel Reaction

Monitoring (PRM)

Metabolite Profiling

(Biocrates)?

Quantitative Proteomics Quantitative Small Molecule

Lipids, drugs, and

other

*Upper mass limitation.

Overview of the Services available to Center Investigators

Identification of Protein and Protein Posttranslational Modifications

Cell Lysate Tissue Biological Fluid

Protein Extraction

Enzymatic Digestion

Cellular Fractionation

Protein Fractionation

Peptide Fractionation

Protein enrichment

Peptide enrichment

1st Dimension Chromatography

2nd Dimension Chromatography

Mass Spectrometry

Data analyses

Subcellular Fractionation

Results Interpretation

Sample Preparation

Data Acquisition

Data Processing

Resource

Users

Optional

400 600 800 1000 1200 1400 1600 1800 2000m/z

b(12)

b(15)b(13)

b(10)

b(14)++

b(19)++

y(13)

y(12)

y(10)y0(17)++

y0(11)++y(10)++

b0(17)++

MATKAVCVLKGDGPVQGIINFE

MS/MS

0 5 10 15 20 25 30 35 40 45 50 55 60 65Time (min)

20.42 24.3335.14

27.96

39.02TIC

YHGHS293MSDPGVS300YR

300 500 700 900 1100 1300 1500 1700m/z

~ 2X

~ 2X

~ 2X

~ 2X

b(3)

b(8)

y(9)

y(6)

y(12)

y(4)

y(6)-98

y(10)-196

y(8)-98

b(2)

y(3)-98

y(2)b(8)-98

b(4)

b(5)-98

b(13)-196

b(11)-98

b(11)y(12)-196

y(11)-196

600 800 1000 1200m/z

782.4141z=3

1173.1164z=2899.9755

z=2

1164.1116z=2

776.4110z=3

1101.5878z=2734.7281

z=3561.6306

z=3*

Start

End

SOP

Quantitative Proteomics Workflow

Enzymatic Digest

Quantitation carried out by additional software:• Progenesis QI Software• Proteome Discoverer• Scaffold Software• Skyline• MASCOT Quantitation

Tool Box

on-line LC MS and LC MS/MS separationand data acquisition

PTM Peptide

enrichment +Flow

through (FT) fraction

Enriched (EN) fraction

Acquired raw Spectral data are processed and detectedfeatures are searched against selected Protein Database using MASCOT/Sequest Search Engine

ReportingTabulated protein list with calculated abundances and expression changes

Biological Source

proteins extraction

SOP

RobustnessStandardized Thoroughness

Intact Protein Analysis Workflow: Determination of Intact Protein MW

Desalting

Desalting image from: http://www.glysci.com/products/Protein%7B47%7DPeptide-Desalting-TopTip.html

Direct Injection ESI MS for data acquisition

MS spectral data are processed and analyzed using Deconvolution Software to obtain accurate high resolution protein mass

Deconvolution of MS to obtain Protein MW

Software for analysis and visualization

New Services coming in 2019…

• Glycosylation workflow.

• Improved LFQ (higher throughput with increased robustness)

• Data Independent Acquisition (improved # of protein identifications)

Cell Lysate Tissue Biological Fluid

Protein Extraction

Enzymatic Digestion

Cellular Fractionation

Protein Fractionation

Peptide Fractionation

Protein enrichment

Peptide enrichment

1st Dimension Chromatography

2nd Dimension Chromatography

Mass Spectrometry

Data analyses

Subcellular Fractionation

Results Interpretation

Sample Preparation

Data Acquisition

Data Processing

Resource

Users

Optional

TechnologyImplementation

Figure 1: Physical appearance of Q-Exactive HF-X mass spectrometer with ACQUITY UPLC M-Class.

New Instrumentations: Improvement in Sensitivity and Throughput

Performance Evaluation of the Q Exactive HF-X for Shotgun ProteomicsChristian D. Kelstrup, Dorte B. Bekker-Jensen, Tabiwang N. Arrey, Alexander Hogrebe, Alexander Harder, and Jesper V. Olsen: Journal of Proteome Research 2018 17 (1), 727-738 DOI: 10.1021/acs.jproteome.7b00602

Future Workflow enhancement for Protein and Protein

Posttranslational Modification Identification & Quantitation

Cell Lysate Tissue Biological Fluid

Protein Extraction

Enzymatic Digestion

Cellular Fractionation

Protein Fractionation

Peptide Fractionation

Protein enrichment

Peptide enrichment

1st Dimension Chromatography

2nd Dimension Chromatography

Mass Spectrometry

Data analyses

Subcellular Fractionation

Results Interpretation

Sample Preparation

Data Acquisition

Data Processing

Resource

Users

Optional

0 5 10 15 20 25 30 35 40 45 50 55 60 65Time (min)

20.42 24.3335.14

27.96

39.02TIC

TechnologyDevelopment

YHGHS293MSDPGVS300YR

300 500 700 900 1100 1300 1500 1700m/z

~ 2X

~ 2X

~ 2X

~ 2X

b(3)

b(8)

y(9)

y(6)

y(12)

y(4)

y(6)-98

y(10)-196

y(8)-98

b(2)

y(3)-98

y(2)b(8)-98

b(4)

b(5)-98

b(13)-196

b(11)-98

b(11)y(12)-196

y(11)-196

400 600 800 1000 1200 1400 1600 1800 2000m/z

b(12)

b(15)b(13)

b(10)

b(14)++

b(19)++

y(13)

y(12)

y(10)y0(17)++

y0(11)++y(10)++

b0(17)++

MATKAVCVLKGDGPVQGIINFE

MS/MS

Progress…

Beckman-Coulter PF2D

Chromatofocusing & NPS-RPLC

2D Protein Separation System

Bruker APEX Qe 9.4TeslaFT-ICR MS System

Accurate mass, Top Down, PTM

AB 4700 MALDI TOF/TOF MS System

DIGE, 1D SDS PAGE, LC-MALDI

AB QSTAR XL ESI QTOF

SystemICAT, MudPIT

Micromass QTOF API MS System

Phosphoprotein Profiling

Micromass MALDI TOF MS

Serum Biomarkers, QC

Micromass QTOF Micro MS System

Lipid ProfilingIntact Protein MWMicromass QTOF API

MS SystemPhosphoprotein

Profiling

Micromass MALDI TOF MS

Serum Biomarkers, QC

MS and Protein Profiling Core: Instrumentations (2006)

Thermo Fisher Scientific nano-UPLC ESI LTQ-Orbitrap ELITE MS systems

Agilent 1200 UPLCAB Sciex 4000 QTRAP

MS system

Mass Spectrometers located within the MS & Proteomics Resource (2019)

ELITE

Thermo Fisher Scientific nano-UPLC ESI Q-Exactive Plus MS systems

Thermo Fisher Scientific nano-UPLC ESI Orbitrap Fusion MS systems

Thermo Fisher Scientific nano-UPLC ESI LTQ-Velos MS systems

For Protein ID For Protein PTM, Profiling, & Quantitation

For Metabolism Separation & Quantitation

For Open Access Usage

Waters UPLC (H-Class)

Q-Exactive HF-X mass spectrometer with ACQUITY UPLC M-Class.

New Instrumentation

Instrumentation

2nd Dimension: Non-Porous-Silica Reversed Phase Chromatography Each pH fraction is run for 20-24 min. with 1 minute fraction collection (UV 214nm);

ProteomeLab PF2D (Beckman-Coulter): 2D Chromatofocusing & non-porous RP-HPLC

Samples are run serially

1st Dimension:Chromatofocusing (pH 8.5 to 4.0) (UV 280nm)

Fractions collected every 0.3 pH unit change

Comparative“gel” and UV

view of samples1 and 2

PI: Geoffrey Chupp, Yale University, Int. Med.

0.2 – 1 mg needed per sample condition

Comparative pI – LC (UV) Protein Profiling before/after Drug Treatment

- Partial pI/UV map - The RP-HPLC profiles illustrate differences in the pI 6.0-6.2 fraction - Both the UV and color coded band depictions of these RP-HPLC profiles

(from Beckman-Coulter)

YPED

#spots picked/analyzed 85#spots with proteins id'd 69#proteins id'd with 1 peptide 0#spots with C5/C3 >= 2 fold difference (all spots)

22

#spots with C5/C3 >= 2 fold difference (proteins Id'd)

18

DIGE Results for Sample: DIGE_GEL409 (12092005)Database – NCBInrSearch Engine - MASCOT

Red – Down regulatedBlue – Up regulatedDark Red/Blue – Protein Id’dLight Red/Blue – Protein not Id’d

March 20th, 2019: Q-Exactive HFX with ACQUITY M-Class

Provide support letter for grant applications

Help with grant write-up materials (budgeting, methodology, aims)

Training and education (from Sample preparation to software use)

In person initial consultation

Free access to licensed Proteomics Software Within the MS & Proteomics Resource At Cushing/Whitney Medical Library (contact Rolando Garcia-

Milian) Write our own grants as a PI (NIH SIG, Pilot, NIH R21)

Developing Technology catering to Users experimental needs

Supporting Users

22

Acknowledgement

MS & Proteomics Group

Rashaun Wilson, PhDJean KanyoWendy Wang

All collaborators and clients

Funding

Instrumentations:NIH SIG S10OD023651-01A1: QE-HFX (PI: Lam)NIH SIG 1S10OD019967-01: UPLC (PI: Lam)NIH SIG 1S10OD018034-01 + YSM: Orbitrap Fusion & Q-Exactive PlusNIH CTSA,UL1 RR024139: 4000 QTRAPNIH SIG, RR031795: LTQ Orbitrap ELITEDonation: LTQ Velos

Collaborations and other support:R01 NS109358-01 (2018; PI:Kahle, Collaborator: Lam)R01 AG057912-02 (2018; PI:Levine, Collaborator: Lam)R01 MH115939-01 (2018; PI:Koleske, Collaborator: Lam)R01 GM102262-01 (2017; PI:Turk, Collaborator: Lam)P30DA018343 (2016; PI’s:Narin & Williams; Discovery Core Director: Lam)

Contact: (Tu) tukiet.lam@yale.eduDirector, Yale/Keck MS & Proteomics ResourceDirector, Discovery Proteomics Core of Yale/NIDA Neuroproteomics ResourceDepartment of Molecular Biophysics & BiochemistryYale University300 George Street, Room G008New Haven, CT 06511Phone (203) 785-5086Fax (203) 737-2638http://keck.med.yale.edu/See my publications in PubMed

Investigators

Support

23

WM Keck Biotechnology Resource LaboratoryMS & Proteomics Resource300 George StreetRoom G001New Haven, CT 06510Phone: (203) 737-2205Email: msproteomics@yale.eduLink: https://medicine.yale.edu/keck/proteomics/

Resource Team Members: TuKiet.LamRashaun.WilsonJean.KanyoWendy.Wang

Contact:

@yale.edu