Automated, High Precision Tryptic
Digestion and SISCAPA-MS
Quantification of Human Plasma
Proteins Using the Agilent Bravo
Automated Liquid Handling Platform
Application NoteAuthors

Abstract

Morteza Razavi, N. Leigh Anderson,

An “addition only” tryptic protein-digestion protocol, followed by multiplexed

Matthew E. Pope, Richard Yip, andSISCAPA peptide enrichment, was developed and automated in 96-well format

Selena S. Larkinon the Agilent Bravo Automated Liquid Handling Platform. These combined

SISCAPA Assay Technologies, Inc.,

protocols, taking as little as 4 hours total, were coupled with high throughput mass

Box 53309, Washington DC 20009,

spectrometry for quantitation of plasma proteins over a wide range of abundance.

USAThe complete workflow was demonstrated for suitability of routine quantitation

Christine A. Miller
Agilent Technologies, Inc.,
5301 Stevens Creek Boulevard,
Santa Clara, CA 95051, USA
Terry W. Pearson
Department of Biochemistry andMicrobiology,
University of Victoria, Victoria,B.C., V8W 3P6, Canadaof plasma protein biomarkers based upon sensitivity, multiplexing capability,scalability, reproducibility, and throughput.

Introduction
Quantitation of proteotypic surrogate
peptides in relation to internal standards
allows quantitative, multiplexed
measurement of proteins using methods
derived from classical isotope-dilution
mass spectrometry (MS) of small
molecules1. Triple quadrupole mass
spectrometer (TQMS) instruments havemeasured light:heavy peptide ratios withCVs of less than 2 %2. Since the precisionof MS measurements depends on thevariability of interfering ion signals (matrixand chromatography effects) as wellas the intensity of peptide ion signals,
achieving low CVs for low abundance
peptides is not trivial and adds to the needfor reproducible digestion. Quantitative

Addition-only tryptic digestion, no SPE

reproducibility of tryptic digestion isessential to support the evolution of mass
spectrometric assays for identificationand quantitation of peptides, and ultimate
measurement of proteins as clinically
relevant biomarkers.

automated workflow, together with
an optimized liquid chromatography
procedure, facilitates multiplexed peptide
quantitation with a 3 minute LC/MS
sample cycle time, significantly increasing
the throughput of protein quantitation.

This Application Note presents thedevelopment of a highly reproducible,automated “addition only” method fortryptic digestion of plasma, followed by
the previously-described bead-based
SISCAPA enrichment3 of target peptides
and quantitation using TQMS. Thismethod offers precision, simplicity ofmultiplexing, generality, and is used
to improve sensitivity and increase
the dynamic range and throughput ofpeptide MS assays. The combined,Materials and Methods

SISCAPA capture, wash, elute

30’

Trypsin30’

Quench
10’

60’

Elute

Wash

Denaturant
plateFigure 1. Protocol Overview: Trypsin digestion, SISCAPA enrichment, LC/MS quantitation.

2

An addition-only method was developed
to enable facile, efficient, and highly
reproducible tryptic digestion of plasma
samples followed by SISCAPA enrichmentof target peptides, using an Agilent Bravo
Automated Liquid Handling Platform(Figure 1).

LC-MRM detection

SISCAPA kit

Alkylating
agent

Hardware

Inj

All steps in the workflow protocol werecarried out using the Bravo AutomatedLiquid Handling Platform with 96-channel
LT disposable tip head and one VarioMag
Teleshake plate shaker (Figure 2). Thesteps were programed for execution using
VWorks software.
The workflow requires use of three
distinct Bravo Automated Liquid Handling
Platform Layouts as shown in Figure 3.

Trypsin digestion (Figure 3A)
A 10 µL solution of pooled human plasma
was added to each well of a 96- wellplate containing a dried denaturation
mix, followed by a 30-minute incubation
at room temperature with vigorous
mixing. The alkylation and diluting agentswere then added to the sample plate,followed by the addition of trypsin at an
enzyme:protein ratio of 1:10. The digest
plate was sealed and manually moved to
an incubator at 37 °C for tryptic digestionof 1 to 5 hours.

Agilent Bravo Automated Liquid Handling Platform deck configuration
12

3Alignment station

Alignment station

Orbital shaking station

45

6

Alignment station

Alignment station

Alignment station

78

9

Alignment station

Alignment station

Alignment station

Figure 2. Agilent Bravo Automated Liquid Handling Platform deck configuration for all methods.

1Tips out

2
Reagent tips

3
Digest plate

Contents: Empty
Plate type: 96 LT 200 µL tips

Contents: Tips
Plate type: 96 LT 200 µL tips

Contents: Dried denaturation mixture
Plate type: 96-well DeepWell plate

4Plasma tips

5Plasma sample plate

6Reagent plate

Contents: Tips
Plate type: 96 LT 200 µL tips

Contents: Plasma samples
Plate type: 96-well PCR plate

Contents: Digestion reagents
Plate type: 8-well LP reagent plate

7
Diluent Tips

8
Diluent plate

9EmptyContents: Tips
Plate type: 96 LT 200 µL tips

Contents: Diluent
Plate type: 8-well LP reagent plate

Figure 3A. Agilent Bravo Automated Liquid Handling Platform deck layout: trypsin digestion.

3Stopping digestion, SIS addition,
SISCAPA target peptide
enrichment (Figure 3B)
During tryptic-digest incubation, thedeck was rearranged as shown in
Figure 3B. The tryptic cleavage reaction
was stopped by adding a stopping agent.
Each sample was spiked with 500 fmol
each of SIS peptides and 0.5–1.0 μg eachof corresponding antipeptide antibodies
conjugated to magnetic beads. The sample
plate containing the antibody‑beadcomplex was vigorously shaken for 1 hour
at 1,000 RPM to capture the peptides.

1Tips out

2
Reagent tips

3
Digest plate

Contents: Empty
Plate type: 96 LT 200 µL tips

Contents: Tips
Plate type: 96 LT 200 µL tips

Contents: Digested plasma
Plate type: 96-well DeepWell plate

4SIS tips

5
SIS reagent plate

6
SISCAPA reagent plate

Contents: Tips
Plate type: 96 LT 200 µL tips

Contents: SIS peptide
Plate type: 96-well PCR plate

Contents: SISCAPA reagents
Plate type: 96-well PCR plate

789

Empty

Empty

Empty

Bead wash and elution (Figure 3C)
The Bravo Automated Liquid Handling
Platform deck was set up a third time,(Figure 3C). Three washes were performed
to remove unbound matrix, with a custom
magnet array designed by SISCAPA
Assay Technologies (Figure 4) used topull the magnetic beads to the sides ofthe wells for the unbound peptide digest
to be removed. The enriched peptides
were released from the beads using acidelution.

Liquid chromatography tandem
mass spectrometry (LC/MS/MS)
Analysis was carried out using an Agilent
1290 Infinity ultra high performance liquid
chromatograph and an Agilent 6490
TQMS (Agilent Technologies, CA). Agilent
MassHunter Workstation Software
was used for both data acquisition andanalysis: MassHunter LC/MS DataAcquisition for Agilent 6400 Series Triple
Quadrupole LC/MS v. B.06.00 was used
for data acquisition while MassHunter
Quantitative Analysis v. B.05.02 andMassHunter Qualitative Analysis
v. B.06.00 were used for data analysis.

Figure 3B. Agilent Bravo Automated Liquid Handling Platform deck layout: stopping digestion, SIS
addition, target peptide enrichment.
1Tips 1

2Elution plate

3Digest plate

Contents: Empty
Plate type: 96 LT 200 µL tips

Contents: Empty
Plate type: 96-well PCR plate

Contents: Digested plasma
Plate type: 96-well DeepWell plate4Clean plate5
Wash/elution stock plate

6
Digest recovery plate

Contents: Empty
Plate type: 96-well DeepWell plate

Contents: Wash/elution buffers
Plate type: 384-well plate

Contents: Empty
Plate type: 96-well DeepWell plate

7Tips 2

8Tips 39SISCAPA magnet plate

Contents: Tips
Plate type: 96 LT 200 µL tips

Contents: Tips
Plate type: 96 LT 200 µL tips

Contents: Magnet plate
Plate type: Magnet array plate

Figure 3C. Agilent Bravo Automated Liquid Handling Platform deck layout: bead wash and elution.

4

Results and Discussion
Sensitivity
The Limit of Detection (LOD) was calculated
as the amount of the SIS peptide below
which the CV was consistently greaterthan 50 % and Limit of Quantitation (LOQ)
as the amount of the SIS peptide below
which the CV was consistently greaterthan 20 %. The performance of four
example peptide assays is summarized inTable 1.

Comparison to a reference method
Figure 4. SISCAPA custom magnet array used for washing of magnetic beads.

Table 1. Agilent Bravo Automated Liquid Handling Platform SISCAPA assay performance data.
Mesothelin

Soluble transferrin
receptor

Protein CinhibitorLPS binding
protein

Measured in 10 µL plasma (fmol)

9.98 (± 0.7)

97.8 (± 3.7)755 (± 105)392 (± 36)

LOD (fmol)

0.99 (± 0.1)

0.72 (± 0.07)

1.5 (± 0.03)

0.89 (± 0.15)

LOQ (fmol)

5.11(± 1.6)

0.72 (± 0.07)

1.5 (± 0.03)

0.89 (± 0.15)

10sTfR (mg/L) as measured by SISCAPA

A 5-level calibrator set for Soluble
Transferrin Receptor (sTfR) was
analyzed using the automated protocol
and the results were compared tothe results of the reference methodprovided with the calibrator set. As
shown in Figure 5, a strong correlation(R2 = 0.998) was observed between theSISCAPA measurement of sTfR and its
measurement by this reference method.
The linear regression model had a slopeof 0.1963 ± 0.0003 and a y-intercept of0.4039 ± 0.0082 at a 95 % confidence
interval.

86y = 0.1963x + 0.4039
R2 = 0.9979

42005010203040sTfR (mg/L) as measured by Roche/Hitachi analyzers

Figure 5. Comparison of sTfR measurements using robotic SISCAPA and a Roche/Hitachi analyzer.

5across four technical replicates. Assay
performance is shown using forward andreverse curves (bottom panel).

50100
Plasma volume (µL)

50
40
30
20

R2 = 0.999

100

100,000

Chicken 1:1Mix Human
Plasma volume (µL)
Meso

Measured analyte(fmol)

050
100
Plasma volume (µL)
sTfRFwd
Rev

00.01
1100
10,000Varying spiked peptide (fmol)

R2 = 0.961

2,000
0050
100
Plasma volume (µL)
PCI

4,800

360

3,600

R2 = 1240

R2 = 0.999

2,400120

1,200

Chicken 1:1Mix HumanPlasma volume (µL)
sTfR

100,000

1PCI

00.01
1100
10,000Varying spiked peptide (fmol)

Figure 6. Scalability of digestion and linearity of peptide responses.

61R2 = 0.992

800
0

3,000

050100
Plasma volume (µL)

150

LPSBP

2,500
2,000
1,500
1,000

R2 = 1500
0

Chicken 1:1Mix Human
Plasma volume (µL)
LPSBP

10,000
1,000

100
10

1,600

100,000

1,000

Fwd
Rev2,400

Chicken 1:1Mix Human
Plasma volume (µL)

10,000

100
100

3,200

150

6,000

1,000

100

4,000

7,200

480

0

6,000

150

600

10,0001,0001720

100,000

10,000100150Meso

R2 = 0.999

250

Measured analyte (fmol)

60

0500

Measured analyte (fmol)

20750

Measured analyte(fmol)

R2 = 0.973

8,000

LPSBP

4,000

Measured analyte (fmol)

Measured analyte (fmol)

40PCI

10,0001,000

60Measured analyte(fmol)

Measured analyte (fmol)
Measured analyte (fmol)

800sTfR

1,250

Measured analyte (fmol)

Meso

100

Forward curves were generated
by titrating synthetic light peptide
from 10,000 fmol to 0 fmol while thecorresponding SIS peptide was kept
constant at 500 fmol. For SIS-varying
reverse curves the SIS peptide wastitrated from 10,000 fmol to 56 amol while

Measured analyte (fmol)

Linearity of peptide recovery is shown
as a function of plasma volume for fourtargets (Figure 6) measured by mixing
equal volumes of pooled human plasma(known to contain the four proteins understudy) with chicken plasma (deficient in
the target peptides). In all cases, linear
responses (R2 > 0.999) were observed

maintaining constant light peptide byspiking 500 fmol in all 12 samples. Thecurves were independently generated
and processed using 10 µL/point ofpooled human plasma in triplicate.
Endogenous levels of the analytes(plateaued part of the forward curve)
can be measured,along with limitsof detection (LOD) and limits ofquantitation (LOQ).

Measured analyte(fmol)

Scalability of digestion andlinearity of peptide responses

Fwd
Rev

00.01
1100
10,000Varying spiked peptide (fmol)

100
101Fwd
Rev

00.01
1100
10,000Varying spiked peptide (fmol)Reproducibility of trypsin digestionand SISCAPA enrichment
To evaluate the precision of theautomated SISCAPA protocol, including
the digestion and peptide enrichment
steps, three sets of 12 samples (N = 36) weredigested and processed independently
on 3 days. Coefficients of variation for
the total workflow (digestion + SISCAPA
peptide enrichment + mass spectrometricanalysis) were consistently less than 5 %, andas low as 2.8 % for proteotypic peptides(Table 2). The coefficients of variation
attributable to the tryptic digestion
step were estimated from 0 % to 2.2% for given targets, with the limiting
factor in SISCAPA assay precision being
MS performance rather than sample
preparation.

Table 2. Precision of Agilent Bravo Automated Liquid Handling Platform digestion and SISCAPA workflow.
Addition-only protocol on Agilent Bravo Automated Liquid Handling Platform –10 µL plasma samples in 96-well plates%CV (n = 12)
Mesothelin

sTransferrin
receptor

LPS binding
protein

MSDay 1

5.10

4.00

2.00

MS

Day 2

5.40

2.80

2.00

MS

Day 3

6.70

1.80

2.60

SISCAPA + MS

Day 1

7.00

3.10

2.10

SISCAPA + MS

Day 2

5.10

2.20

2.60

SISCAPA + MS

Day 3

5.90

2.80

2.50

Digest + SISCAPA + MS

Day 1

6.10

2.60

4.70

Digest + SISCAPA + MS

Day 2

4.60

3.30

5.10

Digest + SISCAPA + MS

Day 3

6.30

3.40

4.70

Throughput
This workflow, implemented on the Bravo
Automated Liquid Handling Platform,
facilitates digestion and enrichment of
96 samples in parallel in as little as 4hours. The near-purity of analyte peptides
enriched by SISCAPA antibody capturemeans the subsequent LC/MS quantitationis reduced to a total of 3 minutes persample. Indeed, it has been demonstrated
elsewhere that SISCAPA‑enrichedpeptides can be quantitated using
RapidFire MS for a cycle time of
approximately 10 seconds per multiplexedsample. The combined throughput of thisautomated process is appropriate forindustrial applications including biomarker
verification and quantitative analysis.

7References
1. Rittenberg, D., Foster, G.L., A newprocedure for quantitative analysis by
isotope dilution with application to
the determination of amino acids andfatty acids. J. Biol. Chem., 1940, 133,
737-774.
2. Anderson, et al. Mass spectrometricquantitation of peptides and proteinsusing Stable Isotope Standards andCapture by Anti-Peptide Antibodies(SISCAPA). J. Proteome Res., 2004, 3,235-244.
3. Anderson, et al. High-Throughput
SISCAPA-Based Peptide Quantitation
Using an Agilent RapidFire
High‑Throughput Mass Spectrometry
System. Agilent Application Note
5991-0380EN

www.agilent.com/chem
For Research Use Only. Not for use in diagnostic
procedures.
This information is subject to change without notice.
© Agilent Technologies, Inc., 2014
Published in the USA, March 4, 2014
5991-4120EN