Automated Plasma Processing for
Quantitative, Targeted LC/MS Analysisof Proteins

Application NoteAuthors

Introduction

Lola Fagbami, Eric Kuhn, Susan E.

Variability in the reproducibility of protein digestion is the major source ofAbbatiello, and Steven A. Carrmeasurement variance in stable isotope dilution, multiple reaction monitoring

Broad Institute of MIT and Harvard

mass spectrometry (SID-MRM-MS), and immuno-MRM-MS (SISCAPA) assays.1

Cambridge, MA USA

This application note describes how the Agilent Bravo Automated Liquid Handling
Platform was used for automated digestion in order to improve the reproducibility
between experimental runs and reduce the labor intensity of this step when large
numbers of samples are evaluated. A Bravo Platform at the Broad Institute wasconﬁ gured for automated reduction, alkylation, and proteolytic digestion of plasma
from human and animal samples to complete this assessment. This applicationnote compares the automated trypsin digestion of plasma in 96-well plates using
the Bravo Platform to manual trypsin digestion in microcentrifuge tubes,
demonstrating comparable analytical results between manual and automatedmethods. Automation on the Bravo Platform allows us to process up to 96 samples
per day (as compared to 24 processed manually), enables parallel reagent addition,and improves reserach lab productivity. A future report will detail our use of theAgilent Bravo in targeted immuno-MRM enrichment.

Experimental
Plasma samples of were spiked with
27 proteins fully labeled with 15N (“heavyproteins”, Argonne National laboratories)
to a concentration of 0.33 pmol/μL. Half ofthe samples were added to a 96 deepwellplate and transferred to the Bravo
Platform for digestion, and half were keptin microfuge tubes for manual processing(Figure 1).
Next, urea was added to a ﬁnal
concentration of 6 M and Tris(2-carboxyethyl) phosphine (TCEP) wasadded to a ﬁnal concentration of 25 mM.
Samples were heated and mixed at
800 rpm, for 30 minutes at 37 °C.
Iodoacetamide (IAA) was then added
to a ﬁnal concentration of 80 mM, andsamples were incubated for 30 minutes atambient temperature, in the dark without
shaking. The Bravo Platform deck wasconﬁgured as shown in Figure 2, and theinstrument was entirely enclosed to avoid
light exposure throughout the reduction,alkylation, and digestion procedure.
Enzymes were prepared at 0.5 μg/μL in
50 mM acetic acid to prevent autolysis

plate at position 6 on an Agilent Peltier
Thermal Station (p/n G5498B#021).
Position 4 and 6 devices were controlled
within the Agilent VWorks Automation
Control software using the MTC Controller(p/n G5498B#015). The matching
microfuge samples were mixed in an
Eppendorf Thermomixer.

and were added to a plate on the Peltier
thermal unit set to 4 °C prior to sample
processing. Enzymes were added directly
to the samples and pH 8.1 was maintained
throughout. Plates were mixed andincubated on the temperature-controlled
shaker unit on the Bravo Platform deck asshown in Figure 2. Bravo Platform deck
positions were conﬁgured as follows:•The sample plate was located at
position 5;•

Pipette tips were located at
positions 1, 2, 3, and 8;•Denaturation, reduction, alkylation,and quenching reagents were in a384-well plate in position 7;•Trizma base was located at
position 9.

Heating and mixing were done on anAgilent Heating Shaking Station(p/n G5498B#009) located at position 4, andenzyme preparations were in a 384-well

Next, mass spectrometry grade lysyl
endoproteinase lys-C (Wako Pure
Chemical Industries, Ltd. p/n 125-05061)
was added to produce an enzyme tosubstrate ratio of 1:50. The samples werethen incubated at 37 °C and mixed at 800rpm. Urea concentration was reduced to~1 M by adding 0.2 M Trizma (pH 8.1).
Next, sequencing grade modiﬁed trypsin(Promega p/n V5111) was added to anenzyme:substrate ratio of 1:100, and thesamples were incubated at 37 °C and mixed
at 800 rpm. After 2 hours, a second aliquotof trypsin was added (E:S of 1:100), andincubation and mixing continued overnight(~15 hours). Enzymatic activity wasquenched by adding 50 % formic acid to aﬁnal concentration of 1%.

Pipette tips

37 °C heater/mixer
Spike in 27
N(U)-labeled proteins

Plasma15n=3, 90 µL plasma/wellTubes

Denature

6 M Urea

Reduce25 mM TCEP

Alkylate

80 mM IAADigest1BravoPlatform

1. Lys-C (1:50) 2h
2. Trypsin (1:100) 2h
3. Trypsin (1:100) 15h42573

Spike in 105
13
C/15N-labeled peptides
DesaltNano-rpLC-MRM-MS
Data analysis

8Oasis HLB 60 mg Plate(positive pressure manifold)

384 wellplate(Urea/TCEP/IPP/Formic acid)

6490 Chip Cube

9Pipette tips
Sample plate

SkylineTrizmaRecovery (PAR)
Precision (CV)Figure 1. Digestion workﬂow performed on theBravo Platform versus liquid handler with alight-tight enclosure on the workbench.

64 °C (lys-C/trypsin)
Figure 2. Deck locations of buffers and pipette tips for automated trypsin digestion using the Agilent
Bravo Platform with gripper (p/n G5523A#003) and a 96LT Disposable-tip Pipette Head(p/n G5498B#042).

2

Following reduction, alkylation, anddigestion, a mixture of the synthetic heavylabeled peptides was added to each well at
a molar concentration equal to the original
heavy (15N) protein spikes. 13C/15N-labeled
peptide standards were added to enable
the monitoring of digestion efﬁciency
and reproducibility as determined bySID-MRM-MS.

PAR and CV values were calculated for
102 of the 105 peptides in the study
to determine recovery and precision,respectively (Table 1) and plotted ashistograms (Figure 3). The medianrecovery of peptides from automateddigestion and manual digestion were both
approximately 50 %, and the histogram

Next, the digested plasma samples weredesalted using Oasis HLB60 plates on
a positive pressure manifold. Desalted
samples were reconstituted in a solution
containing 3 % acetonitrile and
5 % acetic acid (3 % ACN, 5 % HOAc) andcentrifuged for 5 minutes at 5,000 x g. The
supernatant was diluted 1:10 in 3 % ACN,5 % HOAc and transferred to an HPLC
vial. One μL of the sample was injected
onto the Agilent HPLC-Chip with dual
trap/analytical columns and analyzed onan Agilent 6490 Triple Quadrupole LC/MSSystem.

Table 1. Median PAR and CV for 102 peptides (3 transitions/peptide) measured by LC/MS, with MRM,
on an Agilent 6490 Triple Quadrupole LC/MS System.

Results and Discussion
The mass spectrometry data were used todetermine peptide recovery and digestion
reproducibility by calculating the ratio ofeach analyte tryptic peptide (derived bytrypsin digestion of the heavy protein) toits corresponding heavy synthetic peptide
spike. For example, a peak area ratio(PAR) of 0.8 would represent a digestion
recovery of 80 %. Process triplicate
plasma digestions were injected once
on the 6490 Triple Quadrupole LC/MS
System with an HPLC-Chip and analyzed
by dynamic LC-MRM-MS.

Sample processing

Median recovery (PAR)Median precision (CV)Manual (tubes)

50 %

5.9 %

Bravo Platform (plates)

56 %

6.4 %

18A

16
Number of peptides

1412108

Bravo Platform

6Manual420060

102030405060PAR708090100110B

50
40
Count

Between one and ﬁve unique tryptic
peptides were chosen to monitor by
MRM-MS for each of the proteins using a
combination of empirical digestion data,
public database information, and ESP,2an algorithm that aids in the selection ofhigh response peptides for electrospraymass spectrometry. Three transition
ions speciﬁc for each labeled form of
the peptide (one for the syntheticallylabeled C-terminal arginine or lysine
standard, and one for the 15N (U)-labeled
peptide derived from the protein), wereselected and programmed into an MRM
experiment that contained a LC gradientmethod optimized for dynamic MRMacquisition.

representing the range of recoveries
for peptides was also very similar for
automated versus manual digestion(Figure 3A). The median CV for thereproducibility of the process triplicate
digestion was approximately 6 % for bothautomated and manual digestion, and thehistograms were also very similar (Figure 3B).

30
Bravo Platform
Manual20
1000510152025CV (%)

30354045

Figure 3. Histogram plots of (A) recovery as determined by heavy protein to heavy peptide peak area ratio(PAR) and (B) process reproducibility (CV).

3

A plot of the recovery of each peptide
from manual versus automated digestion
shows very strong correlation, indicatingthat manual and automated digestion are
essentially identical with respect topeptide recovery (Figure 4).

1
0.9
0.8
0.7Conclusions

In addition, automated preparation on theBravo Platform enables processing of up
to 96 samples at one time, 4 times thenumber that can be processed manually.
This allows for more samples orreplicates to be completed in a single
experimental run, and, since
all samples are processed in parallel,
there is a reduced risk of variability
between samples. The automatedsolution presented in this applicationnote affords the possibility of increasing
overall research lab productivity andachieving more consistent results, while
freeing scientists from routine, manualtasks.

0.6
Manual PAR

The Bravo Automated Liquid Handling
Platform was used for the automateddigestion of plasma samples for SIDMRM LC/MS analysis to improve thereproducibility between experimental runs
and to reduce the labor intensity of thisstep when large numbers of samples areevaluated. The analytical results achieved
were comparable to those obtained by ahighly trained technician using a manual
method of trypsin digestion in microfugetubes based on PAR and CV values.

0.5
0.4
0.3
0.2
0.1
00

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Bravo Platform PARFigure 4. A high degree of correlation was demonstrated between plasma digestions performed manuallyin tubes and those completed on the Bravo Platform using multiwell plates.

References
1. Kuhn, E., et al. Inter-laboratory
Evaluation of Automated, Multiplexed
Peptide Immunoafﬁnity Enrichment
Coupled to Multiple Reaction
Monitoring Mass Spectrometry for
Quantifying Proteins in Plasma. Mol
Cell Proteomics, 2011, doi:10.1074/
mcp.M111.013854.

2. Fusaro, V.A., et al. Computational
Prediction of High Responding
Peptides for Development of Targeted
Protein Assays by Mass Spectrometry
Nat Biotechnol., 2009, 27(2):190-8.

www.agilent.com/lifesciences/automation
For Research Use Only. Not for use in diagnostic
procedures.
This information is subject to change without notice.
© Agilent Technologies, Inc., 2013
Published in the USA, August 8, 2013
5991-0972EN