what is on the horizon? rapidfire: spe/ms/ms life sciences group · 2016-09-02 · what is on the...

What is on the Horizon? RapidFire: SPE/MS/MS

Life Sciences Group

Can (Jon) Özbal, Ph.D.

Director, RapidFire

November 20, 2012 1

Multi-Step Elution

2

Additional Elution Step

3

Additional Elution Step

4

Programmable Valve Settings

Currently using 2 different protocols:

• 4 state protocol: Standard RapidFire operation

• 5 state protocol:

– Pump 2 is now used to pump a new wash buffer/solvent over the

cartridge as an additional wash step

– Sample must be loaded onto the cartridge with aqueous buffer or binding

is greatly reduced

– Once analytes are bound they can be further washed with a low organic

solution

– Loop is now only washed with low organic, not elution solvent: Could

affect carryover negatively

• Would step elutions be valuable?

November 20, 2012

Confidentiality Label

5

Example: THCCOOH

1. Load Sample

2. Wash with aqueous

3. Wash with 50% MeOH

4. Elute with 100% MeOH

5. Re-equilibrate

November 20, 2012


6

Kinetic Assays on RapidFire

7

Can Add Delays in Sequences

8

Simply add a delay (in seconds) at any point in the sequence

Sample Aspiration

9

AKT Reaction Real-Time Kinetic Plot

y = 3.6922x - 1.9266

R2 = 0.9982

0

20

40

60

80

100

0 5 10 15 20 25 30 35

Time (min)

% c

on

ve

rsio

n

Kinase Assay Kinetic Measurement

Rxn initiated manually on deck in a deep well plate

Same well visited once per minute

Kinetic Enzyme Linearity Measurement

Real-Time Kinetic Data: AKT Kinase

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20 25 30

Incubation Time (min)

Perc

en

t C

on

vers

ion

0.7 nM Enzyme

0.8 nM Enzyme

0.9 nM Enzyme

1.0 nM Enzyme

1.1 nM Enzyme

1.2 nM Enzyme

1.3 nM Enzyme

Reaction initated by multi-channel pipette by ATP addition

7 wells visited sequentially

“Kinetic” Inhibition Curve

Real-time AKT kinetics in the presence of inhibitor

0

20

40

60

80

100

0 5 10 15 20 25 30

Time (min)

Perc

en

t co

nvers

ion [inhib] = 5.0 uM

[inhib] = 4.0 uM

[inhib] = 3.0 uM

[inhib] = 2.0 uM

[inhib] = 1.0 uM

[inhib] = 0 uM

Useful when detecting very weak inhibition or activation

Fast Kinetic Measurements

0

5

10

15

20

25

0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2

Incubation Time (min)

Perc

en

t C

on

vers

ion

3 nM Enzyme

5 nM Enzyme

7 nM Enzyme

“Fast” Kinetic Measurement

• 5 data points obtained in 1 minute

• Challenging to perform with quenched reactions

November 20, 2012


14

S-Nitrosation Various Mutant and Wild-type forms

of Thioredoxin Investigated

November 20, 2012


15

Kinetic Experiments

November 20, 2012


16

Raw Data Deconvoluted Data

S-Nitrosation over Time

Reaction of recombinant TRX with GSNO

Rate of S-nitrosation for TRX proteins

2D-MS with RapidFire

19

Analysis of plasma protein-bound drugs

SEC

LC

MS

Fractionate

(60 sec pools)

Extract

LCMS

1 sample

per

120 sec

Reconstructed

Chromatogram

Protein mixture

SEC

SPE

MS

HTMS

quantitative

measurement

every

12 sec

Reconstructed

Chromatogram

Protein mixture

SEC Fractionation / LCMS 2D HTMS

No Fractionation

UV

detector

2D-RapidFire

Mass

Spectrometry

System

Fraction

Collector

(or waste)

API

Mass

Spec

Chromatography

System

eg:

HPLC or FPLC

syringe pump

(internal std)

Schematic of Instrument

10 20 30 40 50 60 70 80 90 100

-0.5

0.0

0.5

1.0

1.5

2.0 P001 (mg/dl)

Fractions

mg

/dl

SEC of Free P001 Fractionation HTMS

Free P001 (no plasma)

71-76

P001 alone

P001 100 M 2D RF-MS sample every 12 s

2D RF-MS of free P001

B

C

A

A280

Mouse Plasma Size Exclusion Chromatography (SEC)

P001 (0.25mg/ml) incubated in pooled mouse plasma

1 11 21 31 41 51 61 71

0

1

2

3

4

P001

B

C

A

Superose Fractions

P001

(mg

/dl)

SEC Fractionation HTMS

Free P001

Mouse Plasma “A”

P001 100 M

10%

“A”

9%

“B”

81%

“C”

SEC RF MS

Quantitative Proteomics

27

Protein Analysis in High Throughput

Challenge:

• Most proteins co-purify in SPE

– Often protein crash is required in sample prep

– Identification of 1 protein in mixture isn’t feasible

• Proteins have many modifications (methylation, acetylation,

glycosylation, etc.)

– 1 protein ► multiple masses

November 20, 2012


28

Stable Isotope Standards and Capture by Anti-

Peptide Antibodies (SISCAPA)

Solution:

1. Select 1 or 2 proteotypic peptides for protein of interest

2. Generate antibodies against these peptides

3. Digest all proteins in sample (plasma, cell) to peptides

4. Add stable isotope internal standard of peptide(s) of interest

5. Enrich peptides via immunoaffinity

6. Screen by RapidFire

November 20, 2012


29

November 20, 2012


30

Example (University of Victoria)

Specific proteotypic peptides of interest are captured from

digested plasma

Significant protocol optimization work to remove non-specifically

bound peptides

Sample Analysis - Agilent

Originally done by LC-MS

• Advantages

– Chromatographic separation

• Maximizes sensitivity

• Allows multiplexing

• Disadvantages

– Minutes per sample

Can RapidFire speed up the analysis without sacrificing too

many of the advantages of LC?

Testing out the RapidFire –

Sensitivity/Concentration Range

11-point curves (50:1 to 1:50 ratios of light:heavy peptide) were

constructed in formic acid for those 5 different peptides

Agreement between AAA and RapidFire Example RapidFire data

How does RF handle real SISCAPA samples?

The full SISCAPA protocol was used to prepare titration curves

in plasma for the low-abundance protein Mesothelin

Samples were run on the 1290-6490 and the RF300-6490

LC/MS RF/MS Column/Cartridge Zorbax 300 SB-C18, 2.1 x 50 mm A (C4 packing material)

Column Temperature

35 °C Room Temperature

Flow Rate 1.2 mL/min 1.25 mL/min

Mobile Phase A 0.1% formic acid in ultrapure water 0.1% formic acid in ultrapure water

Mobile Phase B 0.1% formic acid in 90% ultrapure acetonitrile

0.1% formic acid in 90% ultrapure acetonitrile

Gradient 10% - 16% B in 1 min, to 22 % B at 1.5 min, 40 % B at 1.85 min, 70% B at 1.9 min, then back to 10 % B from 1.95 min to 3 min

None, wash of 3000 ms, elution of 3000 ms, reequilibration of 500 ms

Total Cycle Time 5.5 min/sample ~10 s/sample

Injection Volume 20 µL 10 µL

SISCAPA with RapidFire

November 20, 2012

Agilent Confidential

35

0.001

0.01

0.1

1

10

0.1 1 10 100 1000Peak A

rea R

ati

o (

Vary

ing

/Co

nsta

nt)

Varying Peptide Spike (fmol)

LPS-BP Titration Curve

Fwd

Rev

R² = 0.998 R² = 0.999

R² = 0.997 R² = 0.994

R² = 0.999

0.01

0.1

1

10

100

0.01 0.1 1 10 100

PCI

TgFSP

TgVIF

LBP

Meso

Mesothelin Titration Curve

0.001

0.01

0.1

1

10

0.1 1 10 100 1000

Vari

ab

le/C

on

sta

nt

Peak A

rea R

ati

o

Spiked Varying Peptide (fmol)

HPLC rev

RF rev

HPLC fwd

RF fwd

How does RF handle real SISCAPA samples?

RapidFire LC

Proteotypic

Mesothelin

Peptide

(estimated

in vivo

concentration

<10 ng/mL)

SISCAPA Multiplexing

November 20, 2012

Agilent Confidential

37

0.0E+0

5.0E+3

1.0E+4

1.5E+4

2.0E+4

2.5E+4

3.0E+4

3.5E+4

4.0E+4

5.9 6 6.1 6.2

Co

un

ts (

cp

s)

Time (min)

Meso light

Meso heavy

PCI light

PCI heavy

LBP light

LBP heavy

TgVIF light

TgVIF heavy

TgFSP light

TgFSP heavy

5 peptide / 10 MRM experiment

Scan speed of the 6490 facilitates heavy complexing

within the ~2 second peak widths of RapidFire

Quantitative Proteomics in Pharma?

• Biomarker validation

• Companion diagnostics

• Cell-based Assays

November 20, 2012


38

Acknowledgements

Agilent Technologies

• Michelle Romm

• Kari Schlicht

• Nikunj Parikh

• Vaughn Miller

• Can Ozbal

November 20, 2012 39

SISCAPA Assay Technologies

• Leigh Anderson

University of Victoria

• Terry Person

• Morteza Razavi

Automating Quantitative

Proteomics

40

Agilent Automation Solutions: AssayMAP

November 20, 2012


41

AssayMAP Bravo Probe Syringe Head

Cartridges

AssayMAP cartridges can be used for

immunoaffinity capture

November 20, 2012


42

Feasibility Assay: Anti-FLAG antibody

1. Bind antibody to

AssayMAP

cartridge

2. Bind analyte to

antibody

3. Wash cartridge

4. Elute analyte

November 20, 2012


43

Purification Step

Volume

(µl)

Flow Rate

(µl/min)

Reagent

Equilibration

50

25

1X PBS

Ligand Loading

50

5

5, 15, 50 µg Anti-

FLAG antibody

Analyte Loading

50

2

7-500nM FLAG-

peptideC

Cartridge Washing

50

25

1X PBS

Elution

50

10

12mM HCl +

100mM NaCl

Effect of Antibody Concentration

November 20, 2012


44

50 15 5

Effect of Reusing AssayMAP cartridges

November 20, 2012


45

FLAG-tagged Peptide in Buffer vs Plasms

Similar linear range and LLOQ seen for FLAG-peptide in trypsin

digested rat serum as PBS buffer: Matrix effect eliminated

November 20, 2012


46

Precision and Accuracy of AssayMAP/RapidFire

November 20, 2012


47

FLAG-PeptideC Accuracy* (%) Precision* (%)

Conc (nM) Intraday (n=4) Intraday (n=4)

7.8 85.3 6.6

15.6 114.2 14.0

31.2 108.6 2.4

62.5 94.2 7.2

125 95.1 8.6

250 105.9 8.1

Sample-to-Data Workflows

48

Encore Liquid Handling Robot

November 20, 2012


49

what is on the horizon? rapidfire: spe/ms/ms life sciences group · 2016-09-02 · what is on the...

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