Workflow Automation for LC/MS:
In-Solution Protein Digestion, Peptide
Cleanup, and Strong Cation-Exchange
Fractionation of Peptides Enabled by
AssayMAP Technology
Application Note
Clinical Research

Authors

Abstract

Jason D. Russell,

Sample preparation for LC/MS interrogation of peptides often comprises multistep
workflows including in-solution protein digestion, peptide cleanup, and peptide
fractionation. This process is usually tailored to a specific application based
upon sample characteristics and the goal of the assay (that is, quantification or
characterization). Automation of sample preparation workflows can increase sample
processing capacity, reduce variability, and eliminate the need for skilled labor toperform repetitive tasks. However, automation is not typically used as a platform forprimary assay development because it is rare that assay developers have the expertise
required to develop complex automation protocols. Instead, assays are typicallydeveloped using wet-bench techniques and adapted to automation with the help ofautomation experts.

Zachary Van Den Heuvel,
Michael Bovee, and Steve Murphy
Agilent Technologies, Inc.
Madison, WI USA

The AssayMAP Peptide Sample Prep solution eliminates the requirement for expertisein automation. Instead, assay developers can focus on the science of the assay enabled
by a simple software-user interface and flexible protocols that give assay developers
full control of key experimental variables. Now, the benefits of scalable, precision
automation can be realized without requiring assay developers, scientists, or technicians
to become automation experts. With the AssayMAP platform, entire workflows canbe developed on the same hardware needed to scale when high-throughput sample
processing is required. This reduces or eliminates the additional time and resources
required to adapt protocols to an automation format. Here we demonstrate a typical
LC/MS workflow for discovery (shotgun) proteomics demonstrating in-solution
digestion, reversed-phase peptide cleanup, and strong cation-exchange fractionation(SCX) of peptides all performed using the AssayMAP Bravo liquid handler. More than
15,000 unique peptide sequences were identified from six SCX fractions generated froman E. coli protein lysate in which 64–67 % of those peptides were identified exclusively
in one of the six fractions through the use of step-wise elution from SCX cartridges
using increasing pH or ionic strength.

Introduction
Sample preparation workflows benefit
from automation by permitting the ability
to scale sample processing to meetthe desired throughput and by relieving
the burdens of repetitive, but critical,
sample handling tasks from skilledlabor. For multistep workflows, thisalso minimizes propagation of error andmaximizes reproducibility by leveraging
automation platforms featuring precision
liquid handling. This is especiallyrelevant for protein and peptide analysis
by LC/MS where errors in multistep
sample preparation workflows are
magnified by the extreme precision andsensitivity of state-of-the-art analytical
instrumentation. However, a significant
challenge arises in automation for sample
preparation; existing workflows are often
adapted to, rather than developed on an
automation platform leading to additional
optimization and resource allocation,
which delays the benefits of workflow
automation. To those not expertly
trained on an automation platform,
automation can be viewed as impracticalfor development and overly rigid. Suchrigidity is prized for fully developed
assays, but it can hinder development
because it requires assay developers tohave expertise in automation, to devote
time to becoming an automation expert,
or require them to rely on an automation
expert to aid in assay development.
True enablement, from the standpointof the assay developer, comes from an
automation platform that permits startto-finish assay development, seamlessly
integrates multistep protocols into linked
workflows, and requires no expertise inautomation allowing them to focus on
the science of the assay rather than on
automation.
Agilent has introduced the AssayMAP
Peptide Sample Prep suite of softwareand microscale cartridges for the
AssayMAP Bravo automation platform
to directly address the needs of assay
developers for LC/MS applications in
the context of workflow automation
for proteomics. At the core of thistechnology lies the precision liquidhandling capability of the AssayMAP
Bravo configured to handle microtiterplates. The AssayMAP Bravo head isequipped with an array of 96 syringeprobes (250 µL) operated throughliquid displacement, which enables
strict flow‑controlled microscalechromatography using disposable
5-µL packed-bed cartridges. Additional
differentiating innovations arose when
automation engineers and LC/MS assay
scientists synergistically developed
modular, flexible protocols for proteomics
applications. These protocols arepresented in a simple, easy‑to‑use
software interface giving assay
developers full control of experimental
parameters without requiring an intimate
knowledge of the underlying automation.

2

This allows wet-bench type assay
development on an automation platform.
AssayMAP protocols were developedfor in-solution protein digestion,
peptide cleanup (desalting), and peptide
fractionation. These protocols were
designed to be operated as stand-alone
modules or they can be mated to build
more complex workflow architectures
facilitated by a host of automation
utilities to transition between protocols.
In this application note, we describe
a multistep AssayMAP workflow for atypical discovery (shotgun) proteomics
experiment whereby E. coli protein
lysate was digested, subjected toreversed‑phase cleanup, and fractionatedusing strong cation-exchange
fractionation with step-wise elution byincreasing ionic strength or pH. LC/MS
analysis using an Agilent 6550 iFunnel
Q-TOF LC/MS system with an Agilent
1290 Infinity LC System and AdvanceBio
Peptide Mapping column identified morethan 15,000 unique peptide sequencesfrom E. coli using either fractionation
technique. Of those 15,000 peptides,
approximately 10,000 peptides wereidentified exclusively in 1-of-the-6
fractions demonstrating the utility ofstep‑wise fractionation from AssayMAP
SCX cartridges. Replicate analyses
revealed the highly reproducible LC/MS
workflow for proteome characterizationby precision Agilent analytics.

Experimental
In-solution digestion

Lyophilized E. coli protein lysate
was reconstituted to 10 µg/µL witha urea‑based Denaturation Mixture(Table 1). Ten-microliter aliquots weremade into each of four wells of two
U-bottom sample plates. The In-Solution
Digestion protocol can accommodate up
to four 96‑well microplates. In this case,all samples could have easily fit onto one
column of a sample plate, but because
the samples were going to be further
processed post-digestion on different
days, it was advantageous to split the
samples onto two plates. Reagent
plates were prepared to match the wellconfiguration of the sample plates.
The In-Solution Digestion protocol was
launched and all plates were placed on
the deck of the AssayMAP Bravo.

Table 1. On-deck samples and reagents for AssayMAP In-Solution Digestion, Peptide Cleanup, andFractionation protocols.
On-deck samples and reagents
In-solution digestion v1.0
SampleE. coli protein lysate (Bio-Rad Laboratores) reconstituted indenaturation mixture to 10 µg/µL

Denaturation mixture

8 M urea with 5 mM TCEP and 150 mM Tris (pH 8)

Alkylant

70 mM iodoacetamide

Diluent mixture

50 mM Tris (pH 8)

Protease

Agilent proteomics grade trypsin (0.33 µg/µL in 50 mM acetic acid)

Wash station
Peptide cleanup v1.1

Deionized water

SampleTFA-acidified tryptic digests of E. coli (pH ~ 2.6)

Stringent syringe wash
buffer (priming)

99.9% ACN/0.1% TFA

Utility buffer(equilibration/wash)

0.1% TFA

Elution buffer

70% ACN/0.1% formic acid

Wash station
Fractionation v1.0

Deionized water

SampleTryptic digests of E. coli in SCX fractionation equilibration buffer

Fractionation method

SCX: Step-wise elution by increasing ionic strength

Priming buffer

5 mM KH2PO4, 350 mM KCl; pH 2.6/25% ACN

Equilibration buffer
Elution Buffer 1

5 mM KH2PO4, pH 2.6/25% ACN
5 mM KH2PO4, 30 mM KCl; pH 2.6/25% ACN

Elution Buffer 2

5 mM KH2PO4, 50 mM KCl; pH 2.6/25% ACN

Elution Buffer 3

5 mM KH2PO4, 85 mM KCl; pH 2.6/25% ACN

Elution Buffer 4

5 mM KH2PO4, 115 mM KCl; pH 2.6/25% ACN

Elution Buffer 5

5 mM KH2PO4, 150 mM KCl; pH 2.6/25% ACN

Elution Buffer 6

5 mM KH2PO4, 350 mM KCl; pH 2.6/25% ACN

Fractionation method

SCX: Step-wise elution by increasing pH

Priming buffer

400 mM ammonium formate, 1% formic acid/25% ACN

Equilibration buffer
Elution Buffer 1

1% formic acid/25% ACN
40 mM ammonium formate, pH 3.5/25% ACN

Elution Buffer 2

40 mM ammonium formate, pH 4.0/25% ACN

Elution Buffer 3

40 mM ammonium acetate, pH 4.5/25% ACN

Elution Buffer 4

40 mM ammonium acetate, pH 5.0/25% ACN

Elution Buffer 5

40 mM ammonium acetate, pH 5.5/25% ACN

Elution Buffer 6

100 mM ammonium hydroxide, pH 9.5/25% ACN

Wash station

Deionized water

Note: All buffers were made as v/v mixtures. The pH of the aqueous component of each buffer wasmeasured and adjusted before the addition of ACN

3

Application Settings were entered in
the In-Solution Digest user interface
using the values indicated in Figure 1
and the protocol was initiated. After the
Denaturation Mixture was added, thetwo sample plates were removed fromthe deck, sealed with a removable plastic
seal using a PlateLoc, and placed in a
60 °C incubator for 1 hour for protein
denaturation and reduction of disulfide
bonds. The sample plate was briefly
centrifuged, the seal removed, and theplate was placed back on the deck.
The protocol continued with the additionof alkylant, an incubation step to alkylate
free cysteines (40 minutes at room
temperature), the addition of diluent, anda trypsin addition (1:33; trypsin:protein).
These were all carried out sequentiallyon deck without interruption or userintervention. After the protocol wascomplete, the sample plates were
removed, sealed using a PlateLoc,
and placed into a 37 °C incubator forovernight digestion. The sample plates
were removed from the incubator andbriefly centrifuged. Each sample plate
was acidified by the addition of 20 µL of
10 % TFA. One plate of acidified E. coli
digests was immediately processed
through the Peptide Cleanup Protocol andthe other plate was stored at –80 °C until
needed. The same digestion experiment
was repeated at a later time generating
two additional sample plates.

pressure to seat AssayMAP cartridges infull-column (eight‑cartridge) increments.
Optimum cartridge performance depends
on proper seating of AssayMAP
cartridges onto the probes of the
AssayMAP Bravo 96-channel head.
AssayMAP Resin-Free cartridges serveas convenient, reusable, substitutes
for packed cartridges when the numberof samples is not a multiple of 8. The
U-bottom sample plate from the digestion
step containing the four wells (A1‑D1) of
TFA-acidified E. coli digest was placed

at deck location 4. The Peptide Cleanup
protocol was launched and Application
Settings were entered on the Peptide
Cleanup user interface, and labware types
were selected in the Labware Table asshown in Figure 2. A volume of 245 µL of
E. coli digest (~ 92.5 µg) was aspirated
onto each AssayMAP C18 cartridge.
Samples were eluted in 20 µL of elution
buffer and diluted 10-fold with theappropriate SCX equilibration buffer.

Figure 1. User interface for the Agilent AssayMAP In-Solution Digestion protocol with the parameters
used for E. coli digestion.

Peptide cleanup

Buffers (Table 1) were dispensed into
the first column of the 12-column plates
corresponding to the reagent plates for
Stringent Syringe Wash (priming) Buffer,
Utility (equilibration/washing) buffer, andElution Buffer. Empty U-bottom plates
were used for Flow Through Collection
and Eluate Collection plates. A totalof four AssayMAP C18 cartridges andfour AssayMAP Resin-Free cartridges
were used to fill a single column in the
Cartridge Seating Station. Although only
four E. coli digests from a single sample
plate were being processed throughPeptide Cleanup, eight cartridges wererequired to fill out a full column for properseating of the AssayMAP cartridges. The
AssayMAP Bravo applies a differential

Figure 2. User interface for the Agilent AssayMAP Peptide Cleanup protocol with the parameters used forcleanup of TFA-acidified E. coli tryptic digests.

4Strong cation-exchange
fractionation by increasing ionicstrength

Under low pH conditions (~ < pH 3),
positively charged peptides can bebound and eluted from negatively
charged strong-cation exchange resinsusing low-pH buffers of increasing ionicstrength (increasing concentration ofsalt). In general, peptides elute from SCX
stationary phases in order of increasing
net positive charge (increasing numberof basic residues)1. The presenceof organic solvent in the buffers(typically acetonitrile) helps to minimize
nonspecific hydrophobic interactions of
peptides with the stationary phase.
SCX elution buffers for step-wise
fractionation using ionic strength(Table 1) were aliquoted into the wellsof U-bottom plates corresponding to
the same well locations of the samples
in the sample plate. The Plate Stacking
protocol was launched to build a stackof six Elution Buffer plates and a stackof six Fraction Collection plates using
the gripper arm of the AssayMAP Bravo.
Additional reagent and waste plates wereplaced on the deck of the AssayMAP
Bravo (Figure 3) as was the sample
plate containing the four E. coli digeststhat had been processed through thePeptide Cleanup protocol and diluted in
SCX equilibration buffer. Four AssayMAP
SCX and four AssayMAP Resin‑Free
cartridges were arranged to create a fullcolumn at the Cartridge Seating Station.
The fractionation protocol was initiatedand upon completion, six 20-µL fractionsof each E. coli digest sample had beencollected. In addition, the flowthrough/wash volume for each sample wasretained to assess peptide binding.
Each set of fractions (24 in total), now
containing relatively high concentrationsof nonvolatile potassium chloride salt,
was diluted 10-fold in 0.1 % TFA to

reduce the concentration of acetonitrile,
consolidated onto a single plate, andagain processed through the Peptide
Cleanup protocol. The relatively salt‑freeflow through/wash samples werelyophilized directly along with the eluatesfrom the fractions processed through thePeptide Cleanup protocol.
This collection of 24 fractions and four
flow through/wash samples represent“Day 1” samples. Day 1 samples wereanalyzed by LC/MS. After data collectionof Day 1 samples was complete, thesecond sample plate from the In-Solution
Digestion protocol (“Day 2” samples) was
processed using the same AssayMAP
workflow and analyzed.

Strong cation-exchange
fractionation by increasing pH

SCX fractionation of peptides can also
be accomplished using AssayMAP SCX
cartridges with step-wise elution using
increasing pH. As with SCX fractionation
using increasing ionic strength, peptides
bind to the stationary phase under low pH

conditions. However, elution is achievedby increasing the pH of the elution buffers
rather than increasing ionic strength. As
the pH is raised during elution, the net
positive charge of peptides decreases.
When the pH of the elution buffer
approaches the isoelectric point (pI) of
peptides, the electrostatic interactions
with the negatively charged stationary
phase weakens facilitating elution2.
Peptides are expected to elute in orderof increasing pI, which also generally
correlates to an increasing number ofbasic residues.
SCX fractionation of peptides using
step‑wise pH elution was carried out
in the same manner as elution by ionicstrength except that a different buffer
system was used (Table 1). However,cleanup of these fractions was not
required because of the use of volatilebuffers that allowed the samples to belyophilized directly. This process resulted
in the collection of 24 fractions and four
flow through/wash samples for each“Day 1” and “Day 2” sample set.

Figure 3. User interface for the Agilent AssayMAP Fractionation protocol with the parameters used for
SCX fractionation of E. coli tryptic digests.

5

LC/MS

All LC/MS analyses were conducted
on an Agilent iFunnel 6550 Q-TOF
LC/MS equipped with a Dual Agilent
Jet Stream ESI source (Table 2). The
mass spectrometer was coupled to a1290 Infinity Binary LC System housing
an AdvanceBio Peptide Mapping C18
column (2.1 × 250 mm, 2.7 µm, 120 Åpore size) maintained at 55 °C. Mobile
phase A was 0.1 % formic acid inwater, and mobile phase B was 99.9 %acetonitrile with 0.1% formic acid. E. coli
fractions were reconstituted in 30 µL
of mobile phase A. Twenty microlitersof each fraction was injected andchromatographed over a 60-minutelinear gradient from 2.5 %–35 % B ata flow rate of 400 µL/min. The Q-TOF
was operated in Auto MS/MS mode inwhich the 10 most abundant precursors
were selected for MS/MS per cycle
using the narrow isolation parameterand an active exclusion time of 1 minute.
The acquisition rate was set at 8 Hz for
MS and 3.5 Hz for MS/MS scans. Aninternal reference mass was not used,
but mass calibration was performed
daily. Data analysis was performed using
a combination of Agilent MassHunter(Version B.05.00, Build 5.0.519.0),
Spectrum Mill (Rev B.04.00.127),Morpheus (64-bit, for Agilent .d files) 3,
and custom scripts.

Table 2. Analytical instrumentation, consumables, and accessories
Analytical instrumentation and consumables
Automation
Core automation platform

Agilent AssayMAP Bravo (G5542A)

AssayMAP starter kits

96 C18 cartridges and labware for digestion and cleanup (G5496-60013)
96 SCX cartridges and labware for fractionation (G5496-60014)
96 RP-S cartridges and labware for digestion and cleanup (G6596-60034)

AssayMAP cartridges(Qty. 96 cartridges/rack)

96 C18 cartridge rack (5190-6532)

AssayMAP Bravo
accessories

Risers, 146 mm (G5498B#055)

96 SCX cartridge rack (5190-6536)
96 Resin-Free cartridge rack (G5496-60009)
96 RP-S cartridge rack (G5496-60033)
Peltier Thermal Station with STC controller (G5498B#035)
Custom Plate Nest (G5498B#017)
PCR Plate Insert (G5498B#013)

Additional accessories

Agilent PlateLoc Thermal Microplate Sealer (G5402A)

LC/MS
Mass spectrometer

Agilent 6550 iFunnel Q-TOF LC/MS

LC System and columns

Agilent 1290 Infinity LC System

Dual Agilent Jet Stream ESI
Agilent AdvanceBio Pepide Mapping Columns (C18)
Analytical: 2.1 × 250 mm, 2.7 µm (651750-902)
Guard:

6

2.1 mm Fast Guard (851725-911)

Results and Discussion
Large-scale SCX fractionationof E. coli digests with an all
AssayMAP workflow

LC/MS results of the multiday
experiments in which E. coli protein
lysates were digested, cleaned up/
desalted, and fractionated using
AssayMAP protocols and cartridges areshown in Table 3. For both step‑wiseelution by increasing ionic strength
or increasing pH, an average of morethan 15,000 distinct peptide sequenceswere identified for each sample
replicate (n = 8) after filtering to a 1 %false discovery rate (FDR) for eachfractionation experiment. Very few
peptides were identified in the sample
flow through/wash demonstrating boththe effectiveness of the Peptide Cleanup
protocol for removing salts and the nearquantitative binding characteristicsof tryptic peptides on AssayMAP
SCX cartridges under the described
experimental conditions.
For SCX by increasing ionic strength,
more than 10,000 distinct peptides
were fractionated in a manner to binthem exclusively into one of the six
fractions. For example, for SCX Fraction 2(50 mM KCl), there was an averageof ~25,750 MS/MS spectra acquired
per 60-minute gradient leading to
9,035 peptide-spectrum matches (PSMs)
at a 1 % FDR. Those PSMs reduced to
6,424 distinct peptide sequences within
Fraction 2. Of those 6,424 distinct peptide
sequences, 3,471 were found exclusively
in Fraction 2.

An aggregate analysis of all of thefractions in a sample set shows that, on
average, 67.1 % of the distinct peptides
were binned into a specific fraction.
This has to be considered within thecontext of typical data‑dependent
MS/MS experiment relying on automatedpeptide‑spectrum matching. However,these data reveal highly efficientstep‑wise fractionation from AssayMAP
SCX cartridges. Similar results wereachieved with step-wise pH elution inwhich 64.2 % of the distinct peptides
were binned into a specific fraction.

Table 3. Peptide identifications from LC/MS analyses of SCX-fractionated E. coli tryptic digests.

E. coli(n = 8) SCX fraction no.

Avg. no.
of PSMs*

Avg. # of distinct
peptides found inspecified fraction

Avg. # of distinct
peptides exclusive
to specified fraction

Avg. % of distinct
peptides exclusive
to specified fraction

Increasing ionic strength
Flow through/wash

117

83

39

47.0 %

Fraction 1 (30 mM KCl)
Fraction 2 (50 mM KCl)

2,853
9,035

1,544
6,424

471
3,471

30.5 %
54.0 %

Fraction 3 (85 mM KCl)

7,692

5,245

1,774

33.8 %

Fraction 4 (115 mM KCl)

9,011

5,424

2,221

40.9 %

Fraction 5 (150 mM KCl)

5,858

3,538

1,055

29.8 %

Fraction 6 (350 mM KCl)

5,711

3,195

1,200

37.6 %

Summary

40,277

15,241‡

10,231

67.1 %

178

109

33

40.4 %

Fraction 2 (pH 4.0)

7,157
11,001

3,678
6,544

1,686
2,933

45.8 %
44.8 %

Fraction 3 (pH 4.5)

8,020

5,083

1,502

29.5 %

Fraction 4 (pH 5.0)

8,977

4,997

1,371

27.4 %

Fraction 5 (pH 5.5)

7,503

4,214

730

17.3 %

Fraction 6 (pH 9.5)

9,023

4,709

1,580

33.6 %

Summary

51,85915,335‡9,846

64.2 %

Increasing pH
Flow through/wash
Fraction 1 (pH 3.5)

* Results filtered to a 1% false discovery rate (FDR), PSM = peptide-spectrum match‡
This value considers the overall number of distinct peptides across all fractions and is not a simple summation of thenumber of distinct peptides found within each fraction.

7

Elution of peptides based their
physiochemical properties and modeof elution trended with predictions.
The average number of basic residues(H, K, R) per peptide increased with eachfraction from 0.99 (Fraction 1) to 2.39
(Fraction 6) for elution with increasingionic strength (Table 4). A smaller
increase was observed for elution withincreasing pH from 1.01 (Fraction 1) to
1.87 (Fraction 2). Similarly, the average pI
trended upward with increasing fraction
number for both elution conditions.
In Figures 4 and 5, the total ion
chromatograms (TICs) generated for eachfraction from each of the eight sample
replicates are overlaid showing the highdegree of similarity for inter- and intraday
analyses. Extracted ion chromatograms(EICs) of select peptides are displayed
at the bottom of Figures 4 and 5. Alleight replicates were overlaid and thepeak area % CVs and mass errors weretabulated. For example, the peak area
% CV for the peptide HVAILGDLQGPK
was 2.8 % with an average mass error of–5.6 ppm without the use of an internal
standard or reference mass correction(Figure 4, bottom). Importantly, thislevel of reproducibility was achieved
after going through an AssayMAP
workflow involving in-solution digestion,
C18 cleanup, SCX fractionation (ionicstrength), and another round of C18
cleanup. Similarly, the peptide VVDAAVEK
had a peak area % CV of 5.3 % with anaverage mass error of 4.9 ppm having
gone through a similar workflow using
SCX fractionation by pH and a single
round of cleanup (Figure 5, bottom).
Peak area % CVs ranged from 2.3–8.1 %with mass errors less than 8 ppm forthe 12 peptides shown at the bottom of
Figures 4 and 5. In addition to low % CV
and mass error, the average retention
time variation for the 12 peptides acrossall eight replicates was less than 0.085 %(< 3 s).

Table 4. The average number of basic amino acid amino residues and estimated isoelectric point (pI) of
distinct peptides identified in E. coli fractions.
Avg. no. of basic
residues (H, K, R)

Estimated*
avg. pI

Flow through/wash

0.21

5.63

Fraction 1 (30 mM KCl)
Fraction 2 (50 mM KCl)

0.99
1.06

6.36
6.72

Fraction 3 (85 mM KCl)

1.48

6.76

Fraction 4 (115 mM KCl)

1.83

6.87

Fraction 5 (150 mM KCl)

2.08

6.94

Fraction 6 (350 mM KCl)

2.39

6.90

Increasing pH
Flow through/wash

0.39

5.54

Fraction 2 (pH 4.0)

1.01
1.09

6.30
6.72

Fraction 3 (pH 4.5)

1.39

6.75

Fraction 4 (pH 5.0)

1.72

6.64

Fraction 5 (pH 5.5)

1.73

6.69

Fraction 6 (pH 9.5)

1.87

6.71

SCX Fraction no.
Increasing ionic strength

Fraction 1 (pH 3.5)

Values were calculated using the “avg. no. of distinct peptides found in specifed fraction” listed inTable 3.
*The isoelectric point (pI) of peptides are simple solution-phase estimates and do not consider theeffects of neighboring residues on pKa values. The pKa values for side chains of H, K, R, D, E, and Y
residues and N- and C-termni were used for simple pI estimates.4

Peptide fractionation can serve
multiple purposes depending on thespecific goals of the experiment. For
discovery‑style experiments (such as
the one described here) fractionation
decreases overall sample complexity by
dividing the complexity among fractions.
This extends the dynamic range of theassay by permitting the identificationof low‑abundance peptides that would
normally be masked in an unfractionatedsample. In general, this increases thenumber of distinct peptides that can be
identified aiding in characterization of thesample. For example, an unfractionatedE. coli digest (12 µg/injection, roughly the8same TIC signal as a fractionated sample)
prepared on the AssayMAP Bravo wasanalyzed seven times using the same
LC/MS method used for the fractionatedsamples. The amount of total LC/MS
acquisition time (7 hours) was equivalent
to analyzing a set of six fractions andthe flow through. The unfractionatedsample produced more peptide-spectrum
matches (58,753), but yielded only 7,678
distinct peptide sequences comparedto the ~15,000 distinct peptides foundwhen fractionation was employed. In
this case, fractionation was extremely
advantageous enabling the identificationof nearly twice as many distinct peptides.

1.6
0.8

Counts (×107)

4.8
4.0
3.2
2.4
1.6
0.8

SCX Fraction 3 (85 mM KCl)

4.0

3.2

Counts (×107)

Counts (×107)

4.0

5.6 SCX Fraction 2 (50 mM KCl)

Day 1, exp 1
Day 1, exp 2
Day 1, exp 3
Day 1, exp 4
Day 2, exp 1
Day 2, exp 2
Day 2, exp 3
Day 2, exp 4

Counts (×107)

2.4

SCX Fraction 1 (30 mM KCl)

2.4
1.6

SCX Fraction 4 (115 mM KCl)

3.2
2.4
1.6

0.8

0.8

4.0 SCX Fraction 5 (150 mM KCl)

4.0 SCX Fraction 6 (350 mM KCl)

3.2

3.2

Counts (×107)

Counts (×107)

3.2

2.4
1.6
0.8

2.4
1.6
0.8

5

10

15

20

25 30 35 40 45
Acquisition time (min)Fraction 1
EIC
m/z 746.946

Fraction 2
EIC
m/z 511.269

37.7 23.8
24.2
24.6
36.9
37.3
AEIVASFERAVAAVNGPIAQALIGKArea % CV = 2.3
Area % CV = 6.9
Dm/z = –1.4 ppm
Dm/z = –8.5 ppm

50

55

60

5

Fraction 3
EIC
m/z 528.777

29.4

29.8
30.2
GYTVSIFNR
Area % CV = 5.1
Dm/z = –7.9 ppm

10

15

20

Fraction 4
EIC
m/z 624.359

27.3
27.7
28.1
HVAILGDLQGPKArea % CV = 2.8
Dm/z = –5.6 ppm

25 30 35 40 45
Acquisition time (min)Fraction 5
EIC
m/z 596.773

50

55

60

Fraction 6
EIC
m/z 565.637

25.2
25.6
26.0 26.8
27.2
27.6
HFDVDAFDAR
HHQTYVTNLNNLIK
Area % CV = 2.3
Area % CV = 7.6
Dm/z = –2.6 ppm
Dm/z = –0.8 ppm

Figure 4. Overlays of eight TICs (n =4, day 1; n = 4, day 2) for each SCX fraction generated by step-wise elution with increasingionic strength (top). Overlays of eight EICs of peptides found in the indicated fractions. Without the use of internal standards orexternal reference masses, peak area % CVs for these peptides are less than 7.6 % with mass errors less than 8 ppm (bottom).

9

4.0
3.2
2.4
1.6

SCX Fraction 1 (pH 3.5)

6.4 SCX Fraction 2 (pH 4.0)

Day 1, exp 1
Day 1, exp 2
Day 1, exp 3
Day 1, exp 4
Day 2, exp 1
Day 2, exp 2
Day 2, exp 3
Day 2, exp 4

5.6Counts (×107)

Counts (×107)

4.8

2.4

6.4

SCX Fraction 3 (pH 4.5)

5.6

SCX Fraction 4 (pH 5.0)

4.8

3.2

Counts (×107)

Counts (×107)

3.2

0.8

2.4
1.6
0.8

4.0
3.2
2.4
1.6
0.8

4.0 SCX Fraction 5 (pH 5.5)

4.0 SCX Fraction 6 (pH 9.5)

3.2

3.2

Counts (×107)

Counts (×107)

4.0

1.6

0.8

4.0

4.8

2.4
1.6

1.6
0.8

0.8
5

10

15

20

Fraction 1
EIC
m/z 415.735

28.2

2.4

28.6
29.0
VVDAAVEKArea % CV = 5.3
Dm/z = 4.9 ppm

25 30 35 40 45
Acquisition time (min)Fraction 2
EIC
m/z 622.348

9.0
9.4
9.8
VEGGVVDLNTLK
Area % CV = 6.9
Dm/z = 1.7 ppm

50

55

60

Fraction 3
EIC
m/z 565.785

33.6
34.0
34.4
NPTIDAWWKArea % CV = 6.0
Dm/z = 6.1 ppm

5

10

15

20

Fraction 4
EIC
m/z 554.827

32.0
32.4
32.8
VDALALITHR
Area % CV = 7.1
Dm/z = –1.6 ppm

25 30 35 40 45
Acquisition time (min)Fraction 5
EIC
m/z 699.333

50

55

60

Fraction 6
EIC
m/z 639.325

26.9
27.3
27.7 29.6
30.0
30.4
EGFYNNTIFHR
FEAYGWHVIR
Area % CV = 8.1
Area % CV = 3.7
Dm/z = 2.1 ppm
Dm/z = –1.3 ppm

Figure 5. Overlays of eight TICs (n =4, day 1; n = 4, day 2) for each SCX fraction generated by step-wise elution with increasing
pH (top). Overlays of eight EICs of peptides found in the indicated fractions. Without the use of internal standards or externalreference masses, peak area % CVs for these peptides are less than 7.6 % with mass errors less than 8 ppm (bottom).

10

These data demonstrate the utility ofstep-wise fractionation in a solid‑phaseextraction (SPE) format despite the factthat peptide fractionation is normally
performed using gradient elution on ahigh-performance liquid chromatography(HPLC) system. The AssayMAP Bravo
is an exceptional liquid handler capableof reproducibly manipulating small
volumes of liquids through mounted
cartridges. However, the AssayMAP
Bravo is not a 96-channel HPLC andstep-wise fractionation with a 5-µL
packed bed cartridge does not offer thesame separation performance relative toperforming offline gradient fractionation
using an HPLC with a comparatively large
packed column.
There are fewer theoretical plates
available in a small cartridge format
relative to a standard‑sized HPLC column
and it is the number of theoreticalplates that largely dictates separation
efficiency5. Traditional SPE formats
involving manual pipetting or gravity/
vacuum-assisted liquid handling areuseful in simple on/off assays where
the goal is to bind analytes, wash away
contaminants, and simultaneously
elute all target analytes. In these cases,
precise volume and flow control are notalways needed for the experiment to be
successful. By contrast, precise volume
and flow control are both critical for
more nuanced techniques like step‑wisefractionation of chemically similar species
because simple on/off chemistry does
not apply. It is difficult to multiplex
sample processing in traditional SPE
formats such that each well, cartridge,
or packed tip receives identical volumesof liquid delivered through the SPEmaterial at the same flow rate leading
to unsatisfactory reproducibility for
multistep fractionation experiments.

The liquid handling properties of the
AssayMAP Bravo enables reproducible
step-wise fractionation in a multiplexable
format stemming from the ability to
precisely control both volume and flowrate through cartridges engineeredto achieve optimal performance formicroscale applications. The AssayMAP
platform is a bridging technology
enabling multiplexed, step-wise
separations in an analytical SPE format
for those applications not requiring HPLC
performance, but still demanding highly
reproducible fractionation on a scalable
sample preparation platform.

Conclusions
Multistep sample preparation workflows
for peptide analysis by LC/MS can be
automated using the AssayMAP Bravo
resulting in highly reproducible in-solution
protein digestion, reversed-phase peptide
cleanup, and strong cation-exchange
fractionation of complex mixturesof peptides. In a proof-of-principle
discovery‑style shotgun experiment,
100 µg samples of protein from a celllysate of E. coli was carried throughmultistep AssayMAP workflows featuring
SCX fractionation with six elution steps
using increasing ionic strength orincreasing pH. Both forms of fractionation
led to the identification of more than
15,000 distinct peptides empowered byan Agilent 6550 iFunnel QTOF LC/MS,an Agilent 1290 Infinity LC System,
and an Agilent AdvanceBio Peptide
Mapping column. Of those 15,000+
distinct peptides, 67.1 % and 64.2 % werebinned exclusively into one of the six
fractions using SCX by ionic strength orpH, respectively. An analysis of selectpeptides over the course of a multiday
experiment revealed peak area % CVs
ranging from 2.3–8.1 % without the useof internal standards. Compared to an

11analysis of unfractionated samples, nearly
twice the number of distinct peptides
was identified using SCX fractionating
demonstrating the use of step-wise
fractionation to increase assay dynamic
range and improve proteome coverage fordiscovery-style experiments.
These experiments establish the easein which multistep sample preparation
workflows can be automated using the
AssayMAP Bravo and its suite of tools
engineered for precision proteomics.
The AssayMAP Peptide Sample Prep
automation solution gives researchers
and assay developers the ability to
rapidly and systematically evaluate
LC/MS sample preparation conditions,chemistries, and methodologies
without having expertise in automation.
Concomitantly, the AssayMAP Bravo
platform enables seamless throughput
scaling once workflows have been
finalized whether the throughput
requirement is a few samples per day,or hundreds. This enablement stems
from the ability to customize protocols
through a simple and intuitive softwareinterface yielding full control overimportant experimental variables tothe user who no longer needs to be
preoccupied with understanding thecomplexities of precision automation.
For those sample preparation protocols
already developed through wet-benchtechniques, assay developers can readily
adapt their protocols to the AssayMAP
platform leveraging the investments
already made in selecting and optimizing
specific chemistries and experimental
conditions. Overall, these data presented
here demonstrates the high degree ofmeasurement precision that can beachieved using Agilent analytics for
peptide analysis by LC/MS facilitated byautomated sample preparation using the
AssayMAP Bravo.

References
1. A.J. Alpert, P.C. Andrews“Cation‑exchange chromatographyof peptides on poly(2-sulfoethyl
aspartamide)-silica” J. Chromatogr.
1988, 443 (0), 85-96
2. J. Dai, C.H. Shieh, Q.H. Sheng,
H. Zhou, R. Zeng “Proteomic analysis
with integrated multiple dimensionalliquid chromatography/mass
spectrometry based on elution of ion
exchange column using pH steps”
Anal. Chem. 2005, 77 (18), 5793-9.
3. C.D. Wenger, J.J. Coon “A proteomics
search algorithm specifically designed
for high-resolution tandem mass
spectra” J. Proteome. Res. 2013.
4. A.L. Lehninger, Principles ofBiochemistry 1982, Worth Publishers,
New York, 1st Edition.
5. C.F. Poole, S.K. Poole “Theory
Meets Practice” In Solid-Phase
Extraction: Principles, Techniques,and Applications, N.J.K. Simpson, Ed.
Marcel Dekker Inc.: New York, 2000.

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., 2013, 2016
Published in the USA, March 16, 2016
5991-3602EN