introduction to protein/peptide quantitation using normal ... three... · introduction to...

Introduction to

Protein/Peptide Quantitation Using Normal Flow LC/MS

High Sensitivity Protein/Peptide

Quantitation enabling Biomarker

Translational Research

Overview

Introduction

• Mass Spectrometry and Proteomics

Increasing Sensitivity

• Low Flow and Normal Flow

• Agilent JetStream and Agilent iFunnel technology

Increasing Robustness

• Reproducible analysis with complex samples

Conclusion

For Research Use Only. Not for use in diagnostic

procedures.

Mass Spectrometry and Proteomics

Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of charged particles

Proteomics is the study of the structure and function of proteins and how they interact within a complex biological system

Since the emergence of many OMICS discovery technologies, thousands of putative biomarkers have been identified and published harkening the increase in translational opportunities for developing new therapeutics destined for the clinics

For Research Use Only. Not for use in

diagnostic procedures.

Agilent 1290-6490 System

Attomole MS Sensitivity

Robustness and Reproducibility



Low-Flow LC/MS Standard-Flow LC/MS

Increasing Sensitivity for Protein Quantitation


procedures.

• Cap- and Nano-Flow LC provide

excellent sensitivity

• Requires more skill to maintain

and use

• On complex samples, may hit

limit of column capacity (75 µm

id columns)

• Sub-2 micron columns provide

outstanding chromatographic

performance (speed, resolution,

loading capacity)

• Rapid, robust analysis

Enhancing Sensitivity for Higher Flow LC Using More Efficient Ionization of Peptides

2 x10

0.2

0.4

0.6

0.8

1.0

Acquisition Time (min) 3 3.5 4 4.5 5 5.5 6 6.5 7

1 2 3 4 5 6 7 AJS normalized response

ESI relative response

MS Inlet

Microflow LC/MS

Nebulizer Heated Sheath Gas

Thermal Gradient Focusing Region

Heat Sink with Active Cooling

Agilent JetStream interface:

• Thermal gradient focusing electrospray

• Usable with flow rates from 10 µL/min and up

• Yields 3-5x increase in sensitivity for peptides


procedures.

JetStream Interface Allows Robust LC/MS with High Sensitivity

1 fmol on-column

2.1 mm

column

0.5 mm

column 575.5937.5

2.1 mm column

300 µL/min

100 amol – 10 pmol

0.5 mm column

17 µL/min

100 amol – 1 pmol


procedures.

•SIX bores

•HALF as long

Six bores, Half the restriction

means…

• 6 times the amount of

atmospheric gas sampled

AND

• 10X the number of ions

sampled over wide mass

range.

But how do we handle all the extra gas molecules?

Compare:

Standard

Capillary is

180mm

6 Bore

Capillary

Enhancing Sensitivity by Increasing Ion Sampling


procedures.

Enhancing Sensitivity by Transmitting More Ions

Samples

more ions

Removes

neutrals

Gives

higher ion

yield


procedures.

Impact of Ion Funnel on Sensitivity

Observed a 5-10x increase in sensitivity

Linear dynamic range on 6460 LC/MS QQQ (std flow):

200 fmol to 25 pmol on-column

Linear dynamic range on 6490 LC/MS QQQ (std flow):

20 amol to 25 pmol on-column 6460 QQQ

6490 QQQ with iFunnel technology

1 fmol on-column

LVNEVTEFAK

575.5 937.5


procedures.

Increasing Robustness: Reproducibility for 110 Injections (10 fmol SIS Peptides and 2.5 µg Plasma Digest On-column)

Protein Response

%RSD

Ret. Time

%RSD

Adiponectin:

IFYNQQNHYDGSTGK 9.8 0.13

Antithrombin-III :

DDLYVSDAFHK 4.7 0.16

Apolipoprotein A-II precursor:

SPELQAEAK 6.7 0.12

Apolipoprotein C-III:

GWVTDGFSSLK 2.3 0.08

Ceruloplasmin :

EYTDASFTNR 9.6 0.14

Heparin cofactor II:

TLEAQLTPR 6.1 0.15

Histidine-rich glycoprotein:

DGYLFQLLR 3.4 0.02

Kininogen-1:

TVGSDTFYSFK 3.3 0.13

L-selectin:

AEIEYLEK 9.5 0.15

Plasminogen:

LFLEPTR 2.2 0.13

Vitamin D-binding protein:

THLPEVFLSK 3.0 0.12

von Willebrand Factor:

ILAGPAGDSNVVK 9.5 0.15

The samples were provided by Derek Smith and Christoph H. Borchers from the UVic-Genome BC Proteomics Centre

2.2% RSD

n=110

4.7% RSD, n=4

7.9% RSD, n=4

12.3% RSD, n=4

Plasminogen LFLEPTR


procedures.

Absolute quantification

of serum apolipoprotein A-I and B

by SID-MRM-MS

in clinical research

Christa Cobbaert, PhD, clinical chemist

Department of Clinical Chemistry and Laboratory Medicine,

Leiden University Medical Center, Leiden, The Netherlands

SID-MRM-MS approach for assay development

of high abundant serum apo A-I and B


procedures.

Serum Desirable specification

CVw CVg I (%) B (%) TE (%)

Apo A-I 6.5 13.4 3.3 3.7 9.1

Apo B 6.9 22.8 3.5 6.0 11.6

CVw; within-subject biological variation, CVg; between-subject biological variation

I; desirable specification for imprecision, B; desirable specification for inaccuracy

TE; desirable specification for allowable total error

Biological variation

Reproducibility of the SID-MRM-MS method

for serum apo A-I and B

CV (%) normal poolserum (NPS 1) CV (%) normal poolserum (NPS 2)

Apo A-I Instrumental Intrarun Interrun Instrumental Intrarun Interrun

AKPAL 0.9 2.9 4.2 1.6 3.3 4.9

VQP 1.7 2.7 4.9 1.5 4.6 7.2

QGLL 5.1 3.2 8.6 3.2 5.6 7.1

VSFL 3.5 4.3 7.8 3.8 5.6 8.2

Apo B

SVSL 3.8 3.1 6.4 5.4 1.0 4.5

FPEV 2.3 0.8 5.2 1.0 3.3 5.6

TEVIP 3.0 1.8 4.2 0.1 1.4 4.6

TGISP 1.0 1.6 4.3 0.7 0.2 5.1

Instrumental = 5 repeated measurements; intrarun = 5 sample work-ups;

interrun = triplicate work-ups on 5 days (N = 15)


procedures.

R² = 0,9594

0,7

1,2

1,7

2,2

0,7 1,2 1,7 2,2

LC

-MS

g/L

ITA g/L

Apo B SVSL

R² = 0,9821

0,7

1,2

1,7

2,2

0,7 1,2 1,7 2,2

LC

-MS

g/L

ITA g/L

Apo B FPEV

R² = 0,9804

0,7

1,2

1,7

2,2

0,7 1,2 1,7 2,2

LC

-MS

g/L

ITA g/L

Apo B TEVIP

R² = 0,9512

0,7

1,2

1,7

2,2

0,7 1,2 1,7 2,2

LC

-MS

g/L

ITA g/L

Apo B TGISP

R² = 0,9621

0,4

0,8

1,2

0,4 0,8 1,2

LC

-MS

g/L

ITA g/L

Apo B SVSL

R² = 0,9814

0,4

0,8

1,2

0,4 0,8 1,2

LC

-MS

g/L

ITA g/L

Apo B FPEV

R² = 0,988

0,4

0,8

1,2

0,4 0,8 1,2

LC

-MS

g/L

ITA g/L

Apo B TEVIP

R² = 0,9846

0,4

0,8

1,2

0,4 0,8 1,2

LC

-MS

g/L

ITA g/L

Apo B TGISP

Method comparison of SID-MRM-LCMS with a clinical

immunoturbidimetric assay for apo B

serum triglycerides:

0.6 - 2.6 mmol/L

serum triglycerides:

4.1 - 17.8 mmol/L

Y = X


procedures.

Conclusions

• LC/MS is a powerful tool in the targeted proteomics workflow

with its superior sensitivity and specificity

• Agilent JetStream and Agilent iFunnel technologies allow you

to achieve highly sensitive analyses using standard-flow

LC/MS

• Standard-flow LC/MS provides excellent robustness and

reproducibility for reliable protein/peptide biomarker

quantitation


procedures.

Acknowledgements

University of Victoria

Derek Smith

Christoph H. Borchers

Leiden University Medical

Centre

Christa Cobbaert

Agilent Technologies

Yanan Yang

Craig Love

Martin Haex

Christine Miller


procedures.

May 4, 2014


procedures.

19

AssayMAP Automated Protein Sample Preparation

Rachel Bolger, Product Manager

April 2014

The LC/MS Influence

May 4, 2014


procedures.

20

Light Chain

Fc

Fab

Glycosylation

site

Disulfide

shuffling

Pyroglutamate

Deamidation/oxidation

Heavy Chain

21

Peptide Mapping Intact molecular

weight Glycan Analysis

A complete menu of automated methods for protein analysis • with method-specific cartridges

• on a single instrument

• with simplified user-interface

The Vision

May 4, 2014


procedures.

AssayMAP Technology Components

May 4, 2014


procedures.

22

Robust &

Reproducible quantitative

chromatographic

separation method

provides highly

reproducible results

Easy to Use Standardized user interfaces enable control

over key assay parameters, are easy to use

and make methods simple to transfer

Precision & Increased

Throughput AssayMAP Bravo Liquid Handling

platform provides precision pipetting

and parallel processing

AssayMAP Technology - Components

Why Chromatography? AssayMAP is like microscale HPLC columns, run 96 at a time, yielding chromatographic-like performance

AssayMAP Cartridge Traditional Pipet Tip

Column

• Single-pass contact (chromatography!)

• Precise, controlled flow rate

• Quantitative binding & elution

• Efficient removal of contaminants

• Recover 100% of capture

• Minimal volume (10 µL elution)

• Multi-pass contact (equilibrium adsorption)

• No flow rate control

• Non-quantitative extraction

• Less efficient removal of contaminants

• Recover < 100 % of capture

• Diluted >10 X

May 4, 2014


procedures.

24

VS

Bat

h

Shower

AssayMAP Cartridge Portfolio

Affinity

Purification Protein A and G

Cartridges

Protein Digestion (protocol only - no

cartridge)

Peptide Clean

Up C18 & RP-S

Cartridge

Fractionation SCX, C18 & RP-

S Cartridge

Workflows: combine individual Apps into powerful workflows

Purify Digest

Cleanup

Peptide Sample Prep

for mass spec analysis

N-glycan Sample Prep

for HPLC, CE or mass spec

analysis

Release and Label

Glycans

Applications (Apps)

Software Designed for Scientists Flexible and intuitive software, out of the box

May 4, 2014


procedures.

26

Workflow-specific

control parameters

Standardized

deck layout

“Pushbutton”

run controls

Complete Spectrum of Scale

Plate

Hub

PlateLoc Centrifuge

AssayMAP Bravo BenchBot Robot

Multidrop Combi

INHECO

Incubator MP

XPeel

May 4, 2014


procedures.

27

AssayMAP Automated Protein Sample Prep Platform

May 4, 2014


procedures.

28

Powerful enrichment due to small volume

Total workflow reproducibility

Dramatically reduces human variability

Standardized interfaces are easy to use

Out-of-the box automation

Integrated workflows

May 4, 2014


procedures.

29

AssayMAP Automated Peptide Sample Preparation Workflow

Rachel Bolger, Product Manager

4/11/2014

Peptide Sample Prep Workflow

May 4, 2014


procedures.

30

Protein Purification App Protein A or Protein G cartridges

May 4, 2014


procedures.

31

May 4, 2014


procedures.

32

Quantitative recovery >95%

Less than 2% CVs

Protein Purification App Quantitative Recovery

30 min/96 samples

Integrates with other workflows

Very reproducible, CVs = <5%

Minimal hands on time

Elution in < 25µl

In-Solution Digestion App Dramatically reduces hands on time

May 4, 2014


procedures.

33

Peptide Cleanup App

Up to 25X concentration factor!

C18 or RPS Reversed Phase cartridges

May 4, 2014


procedures.

34

BSA digestion and cleanup

Person to person reproducibility

Low intra- and interday CVs

Low elution volume = powerful enrichment

May 4, 2014


procedures.

35

Peptide Fractionation App

Simplify peptide samples by stepwise ionic strength or pH elution.

Strong Cation Exchange cartridges

May 4, 2014


procedures.

36

Fractionation – Optimized

May 4, 2014

Confidentiality Label

37

peptides elute mostly in one fraction

Fractionation – Needs More Optimization

May 4, 2014

Confidentiality Label

38

peptides elute across multiple fractions

E. coli fractionation by SCX

Elution by

increasing ionic

strength (KCl)

May 4, 2014


procedures.

39

AssayMAP Automated Peptide Sample Prep

May 4, 2014


procedures.

40

Consistent recovery across mass loads

Powerful enrichment due to:

small elution volume

fractionation

Total workflow reproducibility

Dramatically reduces human variability

Standardized interfaces are easy to use

Out-of-the box automation

Integrated workflows

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