development, significance, and application of standardized kits … · 2016-09-04 ·...

Cambridge Isotope Laboratories, Inc. | isotope.com

Cambridge Isotope Laboratories, Inc. | www.isotope.com

Development,

Significance, and

Application of

Standardized Kits

for Quantitative

Proteomic Studies

Dr. Andrew Percy Senior Applications Chemist - Mass Spectrometry

For Research Use Only. Not for use in diagnostic procedures.


World’s Leading Stable Isotope Company – 30+ yrs, >400 employees

CIL, Tewksbury & Andover, MA, USA

• Headquarters, Tewksbury

• GMP Production Lab, Tewksbury & Andover

• Organic and Process Chem. Lab, Andover

• Acquired MSD Isotope division - 1993

• Spectra Stable Isotopes - 2008

Euriso-Top, Saclay, France

CIS, Xenia, OH, USA Canada Inc., Montreal, Canada

ABX GmbH, Dresden, Germany

Who is Cambridge Isotope Laboratories (CIL)?

• World’s largest: 18O Separation

13C Separation

• Process Chem. Lab

• GMP Production Lab

• Biotech Lab

• Process Chemistry

• Carbohydrates,

Amino Acids, for

Drug Discovery

• 18F PET Precursors

• 68 Ga PET Precursors

• Organic Chem. Lab


• R&D Hot Lab

• CEA Site

• Organic and Process Chem. Lab

• Sales Office


http://www.abx.de/ABX-portal-photo-big.html


Deuterated Solvents Isotopic Gases

18O Water

• Amino Acids

• Carbohydrates

• Gases

• Fatty Acids/Lipids

• Polymers

• Steroids

• Metabolic Tracers

• Alkanes

• Metals

• Custom Synthesis

• Cell Growth Media

• APIs & Intermediates

Research Compounds

Environmental

Contaminant Standards

• Dioxins and Furans

• PCBs

• PAHs

• Pesticides

• Priority Pollutants

• Endocrine Disruptors

• Brominated Flame Retardants

• Chemical Weapons

• Chemical Contaminants

• Metabolites for Human Exposure

• Perfluorinated Compounds

• Personal & Pharmacare

• Urea (GMP)

• Uracil (GMP)

• Phenylalanine (GMP)

• Sodium Bicarbonate (GMP)

• Methacetin

• Aminopyrine

• Caffeine

• Leucine

• Sodium Acetate

• Glucose

• Cholesterol

Breath Test Substrates

& GMP Products

15,000+ products

Breadth of Stable Isotope Products


Quantitative Proteomics – Need for Benchmarking

∙ Evolving trend to quantify analytes in biological samples to address queries in:

∙ systems biology

∙ disease biomarker relevance

∙ Powerful quantitative technique involves the use of MS/MS detection with isotopically labeled standards.

Quantitative data must be precise and accurate.

Assays must be transferable between laboratories and platforms.

∙ QC kits would help improve reproducibility and transferability of MS-based quantitative assays.

∙ Biomarker assessment kits would help expedite their verification and validation.



• Method Development

• Overview

• Target Selection

• Significance of Standards

• Workflow

• Example

• Quantitative Proteomic Kits

• Products

• Utility

• Applications

• Conclusions and Acknowledgements

Outline



Development Workflow

Target Selection

Internal Std Production and

Characterization

Method Optimization

and Development

Quantitation and Assay Evaluation

Parameter Tuning

Transition Screening

Gradient Evaluation

System Suitability

Repeatability

Reproducibility

Accuracy

Linearity

SIS and/or NAT

AAA

CZE

Dissemination

Manuscripts

Newsletters

Assays

Software

Kits

Protein

Peptide

(Abbreviations: SIS = stable isotope-labeled standard; NAT = natural or endogenous)

• What is the intended purpose of the development?

• What are the target analytes?

• What analytical method is desired?

Example Study Questions



Protein Selection

∙ Discovery-based global proteomic profiling.

∙ bottom-up LC-MS/MS (DDA typical operative mode)

∙ Peer-reviewed literature.

∙ abundance levels in target proteome, differential expression

∙ On-line disease databases.

∙ database of differentially expressed proteins in cancer (dbDEPC)

∙ Clinicians, clinical pathologists, and clinical chemists.

∙ Signaling pathways, cellular processes, and metabolic function.

∙ MAPK and AKT pathways – role in cancer development/progression

∙ complement and coagulation cascades – immunity, inflammation



Peptide Selection

General Requirements

∙ Sequence unique within target proteome.

∙ 7-20 residues in length → optimal ionization and fragmentation.

∙ No missed tryptic cleavages (i.e., internal Lys and Arg).

Example Residue-specific Exclusion Criteria

∙ Cys and Met → prone to oxidation.

∙ N-terminal Gln → cyclize to pyroglutamate.

∙ Sequential Pro → low synthesis yield and poor LC resolution.

∙ Asp-Pro → Asp dehydration to a cyclic imide.



Importance of Internal Standards

∙ Provides strong identity evidence for peptide detection.

∙ 13C/15N-labeled standards are chemically identical to unlabeled counterpart and are distinguishable by mass only.

∙ Corrects for ion suppression and matrix effects.

∙ Compensates for inter-run variability of LC-MS platform.

∙ Improves analytical precision.

Kuzyk M.A. et al. Mol. Cell. Proteomics 8, 2009, 1860.


Time Time

Inte

nsity

Inte

nsity

Fibrinogen gamma chain Gelsolin

NAT XIC Transition

SIS XIC Transition


SIS Proteins

Human Apo A-1 Human IGF-1

Labeling: 15N

Concentration: ~2 mg/mL in PBS

pH: 7.4

Purity: >90% (SDS-PAGE)

MW: 30.2 kDa (as SIS protein)

Significance

• Primary component of HDL.

• Promotes cholesterol efflux to liver.

Labeling: 13C/15N-Lys and Arg

Concentration: ~10 µg/mL in water

pH: 7.0

Purity: >90% (SDS-PAGE)

MW: 7.7 kDa (as SIS protein)

Significance

• Key mediator of growth hormone.

• Role in childhood growth and anabolics.

Cox H.D. et al. Clin. Chem. 60, 2014, 541. Hoofnagle A.N. et al. Clin. Chem. 58, 2012, 777.

• BioExpress® cell growth media kits (e.g., mammals and bacteria) for recombinant protein synthesis with 13C and/or 15N labeling.



• Range of isotopically labeled Fmoc protected amino acids for peptide synthesis (e.g., L-Arg-N-FMOC → 13C6, 99%; 15N4, 99%).

SIS Peptide Synthesis

Synthesis

∙ C-terminal 13C/15N-labels

∙ Fmoc protection chemistry

∙ µmol scale

Robotic Peptide Synthesizer (Protein Technologies)

Purification

∙ RP-HPLC-MS

Characterization

∙ MALDI-TOF-MS

∙ Amino Acid Analysis (AAA)

∙ Capillary Zone Electrophoresis (CZE)

∙ 92% average purity for library standards

User Example: Uvic-Genome BC Proteomics Centre (Victoria, BC, Canada)



Sample

Proteolytic Digest

Sample Digest with Labeled Peptides

Concentrated and Desalted Peptides

reduce, alkylate, digest

solid phase extraction

AND/OR

SIS Protein(s)

SIS Peptide(s)

RP-LC-MS/MS

high pH RP-LC

Peptide Fractions

low pH RP-LC-MS/MS

Protein Quantitation

OR

deplete, enrich, or neither

General Workflow with Labeled Standards



Fit-for-Purpose Approach

Tiers defined by method and analytical characterization:

Carr SA et al. Mol. Cell. Proteomics 13, 2014, 907.

Tier Application Standards Accuracy Reproducibility Repeatability

1 Clinical Required Required High High

2 Research Required Not

Required

Moderate-to-

High High

3 Exploratory Not Required Not

Required

Low-to-

Moderate

Moderate-to-

High



SIS Peptides in Practice – Quantitative Proteomics

Agilent’s Infinity 1290 LC

with 6490 QqQ

Tecan Freedom EVO 150

10 kDa MWCO



136-plex Protein Assay Evaluation

Broad Quantitation Range High Separation Efficiency

8.6% CV, on av. (213 peptides)

Percy A.J. et al. Methods 81, 2015, 24.

Reproducible Quantitation

standard reverse curves (n = 3 analytical)



Qualis-SIS Software Tool

Qualis-SIS Capabilities

1. Standard curve generation.

• Assay performance determination.

2. Sample application to curve.

• Sample concentration.

3. Quality assessment:

• green = acceptable

• yellow = caution

• red = unacceptable



Our Protein Quantitation Kits



PeptiQuant™ QC Kits

LC-MS Platform Assessment

∙ Daily

∙ Lyophilized SIS pep in plasma digest.

∙ XIC inspection and performance tracking.

∙ Comprehensive Monthly

∙ Lyophilized SIS pep in plasma digests.

∙ Generate curves and assess accuracy.

Complete Workflow Evaluation

∙ Monthly

∙ Raw plasma, trypsin, and SIS mix.

∙ Generate curves and assess accuracy.

Additionally Supplied

• SOPs and narrative video.

• Performance quality guide.

• LC-MS conditions and parameters.

• Data analysis software (Skyline, Qualis-SIS)

• Reference values.



QC Kit Method Overview

NAT XIC Transition

SIS XIC Transition

For Research Use Only. Not for use in diagnostic

procedures.


Human Plasma QC Kit Panel (35 Proteins)

Concentration Range: 4 orders of magnitude

Protein MW: 7 kDa (Apolipoprotein C-I) to 513 kDa (Apolipoprotein B-100)

Afamin (P43652) Beta-2-glycoprotein I (P02749) Haptoglobin (P00738)

Alpha-1-antichymotrypsin

(P01011) Ceruloplasmin (P00450) Hemopexin (P02790)

Alpha-1B-glycoprotein (P04217) Clusterin (P10909) Heparin cofactor II (P05546)

Alpha-2-antiplasmin (P08697) Coagulation factor XII

(P00748)

Inter-alpha-trypsin inhibitor heavy chain

(P19827)

Angiotensinogen (P01019) Complement C3 (P01024) Kininogen-1 (P01042)

Antithrombin-III (P01008) Complement C4-B (P0C0L5) Plasminogen (P00747)

Apolipoprotein A-I (P02647) Complement component C9

(P02748) Retinol-binding protein 4 (P02753)

Apolipoprotein A-II (P02652) Complement factor B (P00751) Serum albumin (P02768)

Apolipoprotein A-IV (P06727) Complement factor H

(P08603) Transthyretin (P02766)

Apolipoprotein B-100 (P04114) Fibrinogen alpha chain

(P02671) Vitamin D-binding protein (P02774)

Apolipoprotein C-I (P02654) Fibrinogen beta chain

(P02675) Vitronectin (P04004)

Apolipoprotein E (P02649) Gelsolin (P06396) For Research Use Only. Not for use in diagnostic procedures.


Mouse Plasma QC Kit Panel (34 Proteins)

Concentration Range: 4 orders of magnitude

Protein MW: 11 kDa (Apolipoprotein C-IV) to 506 kDa (Apolipoprotein B-100)

Adiponectin (Q60994) Complement C5 (P06684) Interleukin-1 receptor accessory protein

(Q61730)

Antithrombin-III (P32261) Complement component C8

beta chain (Q8BH35) Lumican (P51885)

Apolipoprotein A-IV (P06728) Complement component C8

gamma chain (Q8VCG4)

N-acetylmuramoyl-L-alanine amidase

(Q8VCS0)

Apolipoprotein B-100 (E9Q414) Corticosteroid-binding globulin

(Q06770)

Phosphatidylcholine-sterol acyltransferase

(P16301)

Apolipoprotein C-IV (Q61268) C-reactive protein (P14847) Phospholipid transfer protein (P55065)

Apolipoprotein E (P08226) Fetuin-B (Q9QXC1) Plasma kallikrein (P26262)

Apolipoprotein M (Q9Z1R3) Fibrinogen beta chain

(Q8K0E8) Plasminogen (P20918)

Ceruloplasmin (Q61147) Fibrinogen gamma chain

(Q8VCM7) Platelet glycoprotein Ib alpha chain (O35930)

Clusterin (Q06890) Haptoglobin (Q61646) Serotransferrin (Q921I1)

Coagulation factor XII (Q80YC5) Hemopexin (Q91X72) Transthyretin (P07309)

Complement C3 (P01027) Histidine-rich glycoprotein

(Q9ESB3)

Complement C4-B (P01029) Inter-alpha-trypsin inhibitor

heavy chain H2 (Q61703)



Visualization of Monthly QC Kit

∙ Uses standard curves of SIS peptides in digested plasma.

∙ Qualis-Monitor: Helps monitor changes in LOQs and concentration of endogenous peptides over time.

∙ Can also be used to compare instruments/platforms.



Utility of QC Kits

∙ Kits can reveal procedural errors or instrument deficits.

Example from Recent Large-scale, Inter-lab Study (16 labs, 19 platforms)

Monthly Platform

Performance Kit Results

Percy A.J. et al. EuPA Open Proteomics 8, 2015, 6.

Monthly Workflow

Performance Kit Results

Further Analysis of

Site 6 Curves



PeptiQuant™ Biomarker Assessment Kits (BAKs)

Supplied Prep. Components

∙ Lyophilized SIS peptide mix for spiking into control digest.

∙ Lyophilized SIS peptide mix for spiking into patient digest.

∙ Human plasma and trypsin supplied upon request.

Kit Contents

∙ Detailed user manual.

∙ Acquisition method and worklist (e.g., MassHunter for Agilent).

∙ Data analysis software.

Kit Strategy

∙ Post-digestion addition of SIS mix to reference and user-supplied (n = 20 or 50) samples.



Representative Chromatograms from BAK-76 Analysis

Quantifier transitions shown only for clarity (3 monitored/peptide)

NAT XIC Transition

SIS XIC Transition



BAK-76 Quantitation and Biological Relevance

Concs determined from peptide std curves. Assay’s provide broad linear range necessary to measure

experimental samples.



Application to Experimental Samples

Qualis-SIS provides QA page to aid interpretation. For Research Use Only. Not for use

in diagnostic procedures.


Correlation to Clinical Reference Ranges

Clinical Reference Range

Control Sample

Patient Sample (n = 20) X

• Reference ranges defined by Mayo Medical Clinic and NIH.

Percy AJ et al. Bioanalysis 7, 2015, 2991. For Research Use Only. Not for use



Human Plasma Apo-14 Kit

Representative XICs Proteins (Peptides)

Apo(a) (1) Apo A-I (2) Apo A-II (2) Apo A-IV (3)

Apo B-100 (1) Apo C-I (2) Apo C-II (2) Apo C-IV (1)

Apo D (2) Apo E (4) Apo H (1) Apo J (2)

Apo L1 (3) Apo M (1)

Quantitation Range: 1.6 mg/mL (for Apo A-I) to 7.8 µg/mL (for Apo C-I)

NAT XIC Transition

SIS XIC Transition



Mouse Plasma BAK-81 and QC

Putative Disease Associations of Proteins in BAK-81:

56 CVD, 34 cancer

Property Range

Protein Level Peptide Level

Molecular

Weight (Da) 1697 to 506367 707 to 1698

Hydrophobicity

(GRAVY) -0.97 to 0.09 -2.23 to 1.07

Isoelectric Point 5.03 to 9.37 3.92 to 9.76

Low pH LC RT

(min) N/A 3.5 to 24.3

Physicochemical Properties of Proteins/Peptides in QC Kit

Percy A.J. et al. Proteomics 2016, submitted. For Research Use Only. Not for use



PeptiQuant™ Kit Summary

Current Inventory and Instrument Applicability

Instrument

Kit Type

Human

Plasma

QC

Human

Plasma

BAK-76

Human

Plasma

APO-14

Mouse

Plasma

QC

Mouse

Plasma

BAK-81

6490 (Agilent) x x x x x

6495 (Agilent) x x x x x

QTRAP 4000 (Sciex) x x x x

QTRAP 5500 (Sciex) x

QTRAP 6500 (Sciex) x

Q Exactive (Thermo) x

TSQ Vantage (Thermo) x

Instrument Run Time for BAKs

Control Analysis Sample Analysis

ca. 15 h (6 stds, n = 3) ca. 16 h-40 h For Research Use Only. Not for use in diagnostic procedures.


ProteusQC™ for Bottom-up Proteomics

Kit Benefits

Precise RT calibration.

Assess LC-MS system suitability.

QC digestion efficiency of ApoA-1.

Signal normalization for absolute/relative quantitation.

Reduces experimental cost and improves

data quality.

Developed in collaboration with Dr. Mike MacCoss (UofW).



Conclusion

• CIL is a world leader in the separation and manufacture of stable isotopes and stable isotope-labeled compounds.

• CIL offers isotopically labeled amino acids, resins, and expression systems for SIS production in quantitative proteomics.

• To expedite performance evaluation and protein biomarker assessment, CIL offers a range of PeptiQuant™ kits:

– QC

– BAK

• Newly developed ProteusQC™ has ability to reduce development time, assess data quality, and improve the precision/accuracy of protein quantitation.

for human and mouse plasma



Acknowledgements

University of Washington

Mike MacCoss

James Bollinger

Cambridge Isotope Laboratories

Krista Backiel

Tasha Agreste

Kate Bennitt

Kevin Millis

MRM Proteomics

Christoph Borchers

Agilent Technologies

Christine Miller

Steve Fischer

development, significance, and application of standardized kits … · 2016-09-04 ·...

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