Automated High-Throughput
Analysis of Proteins and Peptides
in Blood-Based Matrices Using
Immunoafﬁnity Puriﬁcation andUltrafast SPE/MS/MS
Application NoteAuthors

Introduction

Michelle V. Romm, Zachary Van Den
Heuvel, and Vaughn P. Miller
Agilent Technologies, Inc.
Wakeﬁeld, MA, USA

Measuring biomarkers in plasma is challenging due to the complexity of thematrix. Currently, immunoafﬁnity puriﬁcation followed by LC/MS/MS isoften employed to isolate and quantify proteins or peptides. However, bothsteps are limited by inefﬁciency; immunoafﬁnity puriﬁcation is labor intensive,
while LC/MS/MS analysis requires minutes per sample. Automation of theimmunoafﬁnity puriﬁcation step, allowing for simultaneous preparation of
96 samples, followed by online SPE/MS/MS analysis at a rate of seconds per
sample could greatly improve the throughput of this workﬂow. We investigated
the feasibility of using such an automated high-throughput workﬂow to analyzeboth proteins and peptides from blood-based matrices.

Experimental
The RapidFire/MS/MS system consistsof the following modules: an AgilentRapidFire 360, an Agilent 6460 Triple
Quadrupole Mass Spectrometer, andMassHunter Qualitative Analysis
B.04.00 and RapidFire Integrator
Software.
The Agilent Bravo Platform for
AssayMAP consisted of the following
modules: the Bravo Workstation for
AssayMAP, AssayMAP Streptavidin(SA-W) Cartridges, and VWorks
Automation Control Software with the
AssayMAP Afﬁnity Puriﬁcation form.
Samples were analyzed at arate < 15 seconds per sample
using the conditions shown in
Table 1. FLAG®-peptideC and its
internal standard were monitored
simultaneously in all experiments.

Chemicals and reagentsThe analyte FLAG-peptideC and its
stable-labeled isotope internal standard
FLAG-peptideC-(13C5, 15N, D8) weresynthesized by American Peptide
Company, Sunnyvale, CA. Rat serum
was purchased from Xenotech, Lenexa,
KS. All other solvents and reagentswere purchased from Sigma-Aldrich,
St. Louis, MO.

Sample preparation
Calibration standards were preparedby spiking buffer or rat serum with
FLAG-peptideC to ﬁnal concentrations
ranging from 7.8 nM to 1 µM. The
samples were then puriﬁed using theTable 1. Bravo Platform for AssayMAP and RapidFire conditions.
Bravo Platform for AssayMAP conditions
Priming

100 µL at 300 µL/min; 1 × PBS (pH = 7.4)

Equilibration

50 µL at 25 µL/min; 1 × PBS (pH = 7.4)

Ligand loading

50 µL at 5 µL/min;
5, 15, or 50 µg biotin anti-FLAG antibody in 1 × PBS (pH = 7.4)

Sample loading

50 µL at 2 µL/min; FLAG-peptideC in 1 x PBS (pH = 7.4), or Rat serum

Sample chase

5 µL at 2 µL/min; 1 × PBS (pH = 7.4)

Washing

50 µL at 25 µL/min; 1 × PBS (pH = 7.4)

ElutionRapidFire conditions

50 µL at 5 µL/min; 12 mM glycine + 100 mM NaCl

Buffer A

Water with 0.1% formic acid; 1.5 mL/min ﬂow rate

Buffer B

90 % acetonitrile with 0.1 % formic acid; 1.25 mL/min ﬂow rate

Injection volume

10 µL

SPE cartridge

Agilent RapidFire cartridge E (reversed-phase C8 chemistry, G9207A)

RF state 1

sip sensor

RF state 27,000 ms

RF state 35,000 ms

RF state 4
Triple quadrupole conditions

500 msGas temperature

250 °CGas ﬂow

11 L/min

Nebulizer

30 psiSheath gas temperature

250 °C

Sheath gas ﬂow

11 L/minNozzle voltage

1,500 V

Capillary voltage

5,500 V
Q1Q3

Dwell

Fragmentor

CECAVIS

1,008.2

1,034.7

50380

20

3

Quantiﬁer

1,004.7

1,290.4

50380

152

Qualiﬁer

1,004.7

1,029.9

50

380

20

3

Agilent Bravo Platform for AssayMAP
with AssayMAP streptavidin (SA-W)
cartridges (p/n G5496-60010) to
immobilize the FLAG antibody thatbinds the analyte of interest. Eluted
samples were diluted 1:1 with MeOH
containing 0.1% formic acid.

2

Data analysis
RapidFire Integrator software was usedfor peak integration. The quantiﬁer ion
AUC of FLAG-peptide C was normalized
by the AUC of the internal standard.
The data was subjected to linear
regression with 1/x weighting.

Results and Discussion

Next, 5 µg of antibody was immobilizedon the SA-W cartridges. A 6-point
serial dilution of FLAG-peptideC in rat
serum was generated. The dilutions
were loaded onto the cartridges,
washed, and eluted. The standard
curves were identical suggesting thatrecovery efﬁciency was unaffected bysample matrix and that no signiﬁcant
ion suppression occurred in the serum
standard curve (Figure 2). The same
cartridges were reused. Even after
multiple uses of the cartridge, data
quality remained unaffected.

50 µg

515 µgProd/IS (AUC)

45 µg

3210050

100

150

200

250300

FLAG-peptideC (nM)

Figure 1. Loading capacity of cartridge.

98PBS
7Rat serum
6Product/IS (AUC)

The loading capacity of the Agilent
AssayMAP SA-W cartridges is 75 µgof biotinylated antibody. The following
amounts of antibody were immobilizedon SA-W cartridges: 5, 15, and 50 μg.
An 8-point serial dilution of FLAGpeptideC in rat serum was generated
for each antibody concentration.
The dilutions were loaded onto thecartridges, washed, and eluted. The
eluted samples were then diluted andrun on the Agilent RapidFire Highthroughput Mass Spectrometry System(RapidFire/MS). Comparable results
were obtained using 5, 15, and 50 μg ofantibody with up to 250 nM of analyte,suggesting it is not necessary tosaturate the cartridges (Figure 1).

6543210050100150
FLAG-peptideC (nM)

Figure 2. Recovery efﬁciency is unaffected by matrix.

3200250300

876Quant/IS (AUC)

Standard curves of FLAG-peptideC
in rat serum were analyzed to obtain
precision and accuracy values on theBravo Platform for AssayMAP coupledwith the RapidFire/MS. The samples
were analyzed in replicates of four.
Standard curves had excellent linearity
within the measured range, with an
R2 value greater than 0.995 (Figure 3).
Accuracies determined were within
15 % and coefﬁcient of variation
values were all less than 15 % forconcentrations within the measuredrange (Table 1).

5R² = 0.9965

43210050

100150200

These results were presented atthe 2012 American Society of Mass
Spectrometry (ASMS) meeting.1

Figure 3. Representative standard curve for FLAG-peptideC spiked into serum.

Conclusions

Table 1. Intraday precision and accuracy for FLAG-peptideC in serum.

Automated, high-throughput
immunoafﬁnity extraction coupledwith ultrafast SPE/MS/MS allows forthe rapid analysis of FLAG-peptideC
in rat serum. Total analysis time fromserum to results for 96 samples isjust over 1 hour. The immunoafﬁnity
cartridges were optimized for efﬁcientuse of capture antibody and could bereused multiple times. Immunoafﬁnity
puriﬁcation showed equivalent results
between samples prepared in PBS
or serum suggesting that recovery
efﬁciency was unaffected by thesample matrix. The quantitation of
FLAG-peptideC across a concentration
range of 7.8 to 250 nM produced
good linearity, accuracy, and precision
results. This methodology may be
useful for the analysis of similar
biomolecules in complex blood-based
matrices.

250300

FLAG-peptideC (nM)

FLAG-peptideC (nM)

Intraday % accuracy (n=4)

Intraday % precision (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

Reference
1. Michelle V. Romm, et al.;Automated High-Throughput
Analysis of Proteins/Peptides inBlood-based Matrices Combining
Immunoafﬁnity Puriﬁcation andUltra-fast SPE/MS/MS. Presented at
the 59th ASMS Conference on Mass
Spectrometry and Allied Topics, 2012,
Vancouver, BC.

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