Automation of a SISCAPA Magnetic
Bead Workflow for Protein Biomarker
Quantitation by Mass Spectrometry
Using the Agilent Bravo AutomatedLiquid Handling Platform
Application NoteAuthors

Abstract

Leigh Anderson
Anderson Forschung Group
Washington, D.C. USA

An automated protocol has been developed implementing SISCAPA immunoaffinity enrichment of biomarker peptides prior to quantitation by mass spectrometry. In this protocol, magnetic beads coated with anti-peptide antibodies bind
specific target peptides from tryptic digests of samples such as plasma and serum,
after which the beads are washed to remove unbound peptides, and the bound,purified peptides are eluted in small volumes for injection into an LC/MS/MSsystem. Internal standards (stable isotope-labeled versions of the same peptides)
allow accurate quantitation. The Agilent Bravo implementation allows SISCAPA
processing of 96 samples in less than 30 minutes.

Matt Pope, Angela Jackson, and Terry
W. Pearson
University of Victoria
Victoria, BC CanadaPeter Werner andChristine Miller
Agilent Technologies, Inc.
Santa Clara, CA USA

Introduction
Mass spectrometry (MS) has become the benchmark technology for analysis ofpeptides based on three important advantages: 1) precise quantitation (particularlywhen used with internal standards); 2) near-absolute structural specificity(based on unique molecular fragmentation patterns); and 3) facile multiplexingof hundreds of measurements in a single analytical run. These advantages canbe exploited in the analysis of “proteotypic” peptides, which occur uniquely ina single protein sequence, to reveal the concentrations of the parent proteinsin proteolytic digests of complex samples such as patient plasma or serum. The
principal limitation of the technology in research on protein biomarkers has been
sensitivity for low-abundance proteins, which may be present at levels 10 ordersof magnitude below the major plasma proteins such as albumin. This problem has
been solved through specific capture of target peptides by purpose-designed antipeptide antibodies (the SISCAPA technology), yielding >100,000-fold enrichmentof target vs albumin peptides, and thus extending the sensitivity of quantitative
mass spectrometry to match that of immunoassays. When implemented using
established affinity carriers such as magnetic beads, large numbers of samples
can be processed with efficiency and precision. As a result, the inherent quality
advantages of MS measurement can be obtained over the full range of existing
and candidate proteins biomarkers.

Materials

Protocol Workflow• Agilent Bravo Automated Liquid
Handling Platform (G5409A)

The SISCAPA protocol described here
begins with tryptic digests of plasma(or serum) samples to which the twoSISCAPA reagents (antibody-coatedmagnetic beads and stable isotopelabeled peptides) have been added andincubated for 3-15 hours. In typicalpractice, the tryptic digestion has been
carried out in advance, the resulting
tryptic peptides cleansed by solid
phase extraction (SPE), e.g., on an
Oasis plate, lyophilized, and reconstituted in Wash Solution (PBS + 0.03%
CHAPS + 0.02% NaN3, pH 7.4).

• 96-channel LT Disposable Tip Head(04730-202)• VarioMag Teleshake Plate Shaker(on Bravo deck)• 24-pin magnet array(e.g., Novagen Cat. No. 71101-3)• 96-well, polypropylene conicalbottom (V-bottom) plate, having
450 µL capacity per well(e.g., Nalge/Nunc 249944) forprocessing capture reactions• 96-well, polypropylene skirted PCR
plate for SISCAPA-enriched peptides(e.g., Axygen Cat. No. PCR-96-FS-C)• 96-deepwell, polypropylene conicalbottom (V-bottom) plate, having
2 mL capacity (e.g., Geiner 780280)• 384-deepwell, polypropylene
conical bottom (V-bottom) plate,having 200 µL capacity(e.g., Greiner 781271)

In the following Bravo protocol, thebead wash/elution process is implemented as two sequential scripts(A and B) written using the VWorks
software. In script A, stock solutions
provided in rows of a 96-deepwellplate are distributed into wells of
two 384-deepwell plates in order toprovide five separately-accessible setsof 96-wells in two deck positions foruse in protocol B (Table 1, Figure 1).
After script A is complete, the tips
and stock solution plate are removed,
leaving the two 384-deepwell plates indeck positions 5 and 8, and additional
labware is mounted (Table 1, Figure 2).
The SISCAPA reaction plate (in which
each well contains a sample digest,
antibody-coated magnetic beads andadded heavy standard peptides) is
placed on a plate shaker mounted atdeck position 3. Script B then carries
out the following general process:1. Separation of beads from digest
using magnetic methods
2. Three sequential washes of beads
in 150 µL fresh Wash Buffer withminimal carryover volume
3. Elution of bound peptides in 20 µL
low-pH Elution Buffer
4. Collection the beads at neutral pH
for potential re-use• Agilent 96LT 200 μL sterile,filtered pipette tips (08585-102)

Table 1: Bravo Deck Setups• Agilent VWorks Automation
Control software

1 (empty)

Tips 1 (New)

2 (empty)

SISCAPA Eluate (=Output)

3 (empty)

SISCAPA Reaction (=Input)

4 96-Deepwell Stock Solutions

Clean 96-well Washplate

5 384-Deepwell Wash 1-3 & Eluent

384-Deepwell Wash 1-3 & Eluent

6 Tips (Used)

Save Digest Plate

7 Tips (New)

Tips 2 (New)8 384-Deepwell Bead Recovery

384-Deepwell Bead Recovery

9 (magnet plate)(magnet plate)• SISCAPA antibodies covalently
coupled to Protein G magnetic beads(e.g., Dynal Dynabeads G)• Stable isotope-labeled versions oftarget peptides at 100 fmol/µL• Wash Solution: PBS with 0.03%
CHAPS detergent and 0.02% NaN3,
pH 7.4

Plate Prep (Script A)• Elution Solution: 5% acetic acid2

Wash/Elute (Script B)

In order to minimize carryover of
non-specifically bound peptides on
surfaces of plate wells or pipettetips, two sets of tips are used: one“clean” for introducing fresh solutions and collecting the final eluate,
and one “dirty” used to remove digest
and used wash solutions. In addition, the bead suspension is moved
during the second wash cycle fromthe original SISCAPA Reaction plate
to a Clean Washplate prior to the final
wash and elution. During each cycleof magnetic separation, the platecontaining the beads is moved by
the Bravo arm onto a magnetic plate
causing the beads to clump to one sideof the well, after which all the freeliquid can be removed by aspiratingfrom the bottom, center of the well.
Subsequently fresh liquid is dispensed
into the well, and the plate moved backto the shaker, which resuspends thebeads in the fresh solution (1,000 rpm
for 60 sec). Script B requires 20 min,in which the time between removalof beads from sample digest and final
peptide elution is less than 13 min.

Figure 1. Bravo Deck Layout for Script A

The eluted peptides are delivered in a
volume (20 µL) and solvent (5% aceticacid) suitable for subsequent injection into a reversed-phase LC system
feeding a triple quadrupole MS, inwhich multiple reaction monitoring(MRM) methods are used to quantitate
the stable isotope-labeled (“heavy”internal standard) and sample-derived(“light” analyte) version of each
peptide. Analyte concentration iscalculated as the peak area ratio oflight/heavy MRM’s multiplied by theknown concentration of the added
heavy peptide (so-called stable isotope
dilution method).

Figure 2. Bravo Deck Layout for Script B

3

Results and Conclusion
A multiplex SISCAPA experiment
was carried out using 1 µg of eachof 11 rabbit monoclonal antibodies
covalently linked to magnetic beads
to capture 11 respective target
peptides and cognate stable isotopelabeled standards from digests of
10 µL samples of human plasma. The
captured and eluted peptides wereanalyzed using reversed-phase (C18)chromatography at 400 µL/min anddelivered to an Agilent 6490 Triple
Quadrupole LC/MS with iFunnel
technology. Three separate peptide
fragments (transitions) were measured for each peptide and its labeled
cognate (66 MRM channels). The LC
peak shape and elution times of thepeptides are shown in Figure 3.

Figure 3. Peaks in the LC chromatogram produced by the best transition for each of the 11
peptides monitored.

Figure 4 shows example standard
curves obtained by adding varying
amounts of either the heavy internal
standard peptide (“Reverse” curve:
3-fold dilutions down from 400 fmol
per reaction) or a synthetic versionof the endogenous (unlabeled, light)
peptide (“Forward” or standard addition curve). The difference between
the curves is the presence of endogenous light peptide derived from thesoluble transferrin receptor (sTfR)
protein in the plasma sample digested
to provide the analytical matrix.
The results demonstrate that theAgilent Bravo Automated Liquid
Handling Platform can implement theSISCAPA peptide enrichment method,
generating samples suitable for
LC/MS/MS analysis.
www.agilent.com/lifesciences/automaton
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use in diagnostic procedures. Information, descriptions, and specifications in this publication are subject
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Agilent Technologies shall not be liable for errors contained
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© Agilent Technologies, Inc. 2011
Published in the U.S.A., January xx, 20115990-7360EN

Figure 4. Standard curves showing quantitative MS results (ratio of light and heavy
peptide MRM peak areas) for a peptide (GFVEPDHYVVVGAQR - unique to the circulating
fragment of human transferrin receptor sTfR) captured by a rabbit monoclonal antibody
from a tryptic digest of 10 µL human plasma. In the Reverse curve (red), varying amounts
of Heavy internal standard peptide (eleven 3-fold dilutions down from 400 fmol) havebeen added to the digest, yielding a curve decreasing downward to a plateau at the lower
limit of detection. In the Forward curve (blue), varying amounts of Light synthetic target
peptide (eleven 3-fold dilutions down from 400 fmol) have been added to the digest, alongwith a constant amount (50 fmol) of heavy peptide. In this case, the level of the endogenous peptide (250 fmol) present in the sample digest is evident.