Application Note
Biopharma

Automated MHC-Associated Peptide
Enrichment for Immunopeptidomics
Analysis Using Agilent AssayMAP
Bravo Large Capacity Cartridges
Authors
Samuel Pollock
Genentech,South San Francisco, CA, USA
Shuai Wu, Jerry Han, andSteve Murphy
Agilent Technologies Inc.,
Santa Clara, CA, USA

Abstract
This application note describes a method for automated MHC-I associated peptide
enrichment using the Agilent AssayMAP Bravo platform with large-capacity (25 µL)
cartridges for immunopeptidomics analysis. For accompanying LC/MS analysis,an Agilent 6550 iFunnel Q-TOF LC/MS was enabled for nanoflow operation using
an Agilent Infinity UHPLC Nanodapter. This workflow provides a reproducible,
straightforward means of enrichment for MHC-I peptide analysis.

Introduction
When there is a viral infection, cytotoxic
T lymphocytes (CTLs) are activated after
recognizing host cells presenting viral
antigens via the MHC-I display system.
These antigens are generated after viralproteins are degraded into peptides
in the cytoplasm and transported
to the endoplasmic reticulum (ER),where they are loaded into class I
major histocompatibility complexes(MHC-I), which then migrate to the cellsurface. These MHC-I complexes areof interest not only in viral defense,
but also in cancer immunotherapy,
as CTLs can also recognize peptides
derived from endogenous, mutatedproteins.1,2 Therefore, characterizationof MHC-I presented peptides, orimmunopeptidome, is of greatinterest in the design of vaccines andimmunotherapies against cancer andinfectious diseases.3

Immunopeptidomics is generally
considered more challenging in part due
to the fact that most MHC-associatedpeptides are extremely low in abundancecompared to other cellular peptides.
One way to address this challenge is toimmunoprecipitate the MHC-I complexes
from large amounts of cell lysate using
an anti-MHC-I antibody coupled to a solidphase. The MHC-associated peptides
are then isolated from the complex and
identified by mass spectrometry.1–3
High-quality sample preparation is
critical for obtaining good peptide signal
and highly reproducible results. This
workflow used the AssayMAP Bravo
platform for automated immunoaffinity
purification and peptide cleanup. The
AssayMAP Bravo platform is composedof three main components (Figure 1): a
high-precision liquid handler fitted withprobe syringes that provides exquisite
flow control; cartridges with a packed

resin bed that connect to the probesyringes to allow positive flow over theresin bed; and user-friendly softwarewith applications optimized to give
great results straight out of the box withminimal optimization.
In addition to high-quality sample
preparation, a very sensitive andreproducible LC/MS system is required.
For this study, an Infinity UHPLC
Nanodapter converted standard LC flow
to nanoflow. The nanoflow LC systemwas then coupled with a 6550 iFunnel
Q-TOF LC/MS using data-dependentacquisition (DDA) for peptide analysis.
The combination of high-quality sample
preparation and a sensitive LC/MSsystem provide a reproducible andscalable solution to meet the challengesof studying MHC-I peptidomics.

5 µL

Figure 1. Agilent AssayMAP Bravo platform with new AssayMAP 25 µL PAW cartridge (right).

225 µLExperimental

Antibody immobilization andcrosslinking to 25 µL protein Acartridge

Material•

Anti-human MHC-I antibody (w6/32)
was expressed and purified atGenentech.

•The GRANTA-519 cell line wasobtained from Genentech’s cell bank.

•AssayMAP 25 µL Protein A cartridge(PAW) and 5 µL C18 cartridges arefrom Agilent Technologies, Inc.
(Santa Clara, CA, USA) (Figure 1).

•All other chemicals were orderedfrom Sigma-Aldrich (St. Louis, MO,USA).

Anti-human MHC-I antibodies wereimmobilized on 25 µL protein A (PAW)
cartridges using the Affinity Purification
software application on the AssayMAP
Bravo (see Table 1, Immobilization
column, for details). Briefly, phosphatebuffered saline (PBS) pH = 7.4 was used
to prime and equilibrate AssayMAP 25 µL
PAW cartridges. Six of these cartridges
were then loaded in parallel with 1 mg
(1 mg/mL, 1,000 µL) of anti-human
MHC-I antibody (w6/32) per cartridge
using a loading flow rate of 20 µL/min.
Lastly, the cartridges were washed oncewith 250 µL PBS (pH = 7.4).
The PAW cartridges with a resin bed
containing anti-MHC-I antibodies
crosslinked to protein A resin weregenerated by treating the cartridges as
described above, with a crosslinking
reagent using the Affinity Purification

application on the AssayMAP Bravo(see Table 1, crosslinking column, fordetails). Briefly, the 25 µL anti-MHC-I
cartridges were primed and equilibrated
with 0.2 M triethanolamine (pH = 8.1).
Dimethyl pimelimidate (DMP) was used
to crosslink the antibody to protein
A by flowing 200 µL of 5 mM DMP in
0.2 M TEA over the cartridge beds witha loading flow rate of 5 µL/min. The
crosslinking reaction occurred at room
temperature during the 40-minutes
loading. Unreacted DMP was washedaway with TBS and Tris buffer, andnon-crosslinked antibody was washedaway using 1% acetic acid. The newly
crosslinked cartridges were thenequilibrated in TBS buffer (pH = 7.4) andstored at 4 °C until use. The chimneys on
the cartridge plate where the crosslinked
cartridges were stored were filled in withTBS buffer (pH = 7.4) + 0.025% sodium
azide as storage buffer. About 190 µL
buffer was added in each chimney.

Table 1. Affinity purification and peptide cleanup protocol using the Agilent AssayMAP Bravo platform.
Immobilization

Crosslinking

Immunoaffinity PurificationPeptide Cleanup

Resin Bed Volume

25 µL

25 µL

25 µL

5 µL

Affinity Medium

Protein AProtein A + antibody

Protein A + X-linked antibody

C18PBS, pH = 7.4

0.2 M triethanolamine, pH = 8.1

TBS, pH = 7.4

70% ACN/0.1% TFA in water

Prime Buffer
Equilibration Buffer

PBS, pH = 7.4

0.2 M triethanolamine, pH = 8.1

TBS, pH = 7.4

2% ACN/0.1% TFA in water

Loading Buffer

Antibody storage buffer

5mM DMP in 0.2M TEA

3 mg/mL GRANTA lysate

1% Acetic acid

Loading Volume

1,000 µL

200 µL

1,000 µL

200 µL

Loading Flow Rate

20 µL/min

5 µL/min

20 µL/min

5 µL/minWashing Buffer 1PBS, pH = 7.4

TBS, pH = 7.4

TBS, pH = 7.4

2% ACN/0.1% TFA in water

Washing Volume 150 µL

250 µL250 µL250 µLNo. Washes 11111Washing Buffer 2

NA25 mM Tris, pH = 8.0

25 mM Tris, pH = 8.0

NAWashing Volume 2

NA250 µL

250 µLNA

No. Washes 2NA111

Elution Buffer

NA1% acetic acid1% acetic acid

30% ACN/0.1% TFA in waterElution Volume

NA50 µL

50 µL

50 µL3Immunoaffinity purification of MHC-Icomplex
GRANTA-519 cell pellets were lysedin nondenaturing buffer as previously
described.5 The Affinity Purification
software application on the AssayMAP
Bravo was used to purify MHC-I
complexes from the cell lysate (seeTable 1, Immunoaffinity purification
column, for details). Briefly, TBS buffer(pH = 7.4) was used to prime andequilibrate the antibody crosslinked PAW
cartridges. The MHC-I complexes wereimmuno-captured with the antibody
crosslinked cartridges. About 3 mg
(3 mg/mL, 1 mL) GRANTA lysate wasloaded onto each of six cartridges witha loading flow rate of 20 µL/min. Thiswas followed by two sequential wash
steps. The first wash was a 250 µL washwith TBS buffer (pH = 7.4) followed bya 250 µL wash with 25 mM Tris buffer(pH = 8). Lastly, the enriched MHC
complexes were eluted with 50 µLof 1% acetic acid. The eluate fromsix cartridges was combined to generatesample 1 (Table 1, Figure 2). The same
experiment was repeated on differentdays to generate samples 2 and 3.

MHC-I peptide cleanupThe MHC-I peptides were separated
from MHC protein and desalted on C18cartridges using the Peptide Cleanup
application software on the AssayMAP
Bravo system (see Table 1, Peptide

Cleanup column, for details). Briefly,
70% ACN/0.1% TFA in water was used toprime the cartridge. The cartridge wasthen equilibrated with 2% ACN/0.1% TFA
in water. The MHC complex in 200 µL
of 1% acetic acid was loaded on theC18 cartridge with a loading flow rateof 5 µL/min. Cartridges were washedonce with 50 µL of 2% ACN/0.1% TFA inwater. The MHC-I peptides were eluted
with 50 µL of 30% ACN/0.1% TFA inwater (Table 1, Figure 2). Samples werecombined as appropriate and dried downusing Speed-Vac at room temperature.
The dried samples were stored at –80 °C
until analysis.

GRANTA-519
cell lysate

Anti-human MHC-I
antibody (w6/32)
1) Crosslink mAb to PAW
cartridge using DMP

2) Immunoaffinity
purification

3) Combineeluate4) MHC - I peptide
cleanup

5 µL C18
cartridge
25 µL PAW cartridges
Figure 2. MHC-I peptide enrichment and cleanup using the Agilent AssayMAP Bravo platform.

4

Peptide identification using DDA
The dried samples were resuspended in
5 μL of 10% ACN/0.1% FA. The sample
vials were vortexed and sonicated fortwo minutes. Samples were further
diluted in 5 μL of 0.1% FA such that thefinal samples were suspended in 10 µLof 5% ACN/0.1% FA.
An Agilent 1290 Infinity II LC wasconverted to a nanoflow LC by couplingwith the Agilent Infinity UHPLC
Nanodapter. This nanoflow LC wasconnected to the Agilent nanospraysource and coupled with the Agilent
6550 iFunnel Q-TOF LC/MS for peptideidentification (Figure 3). The LC
parameters were listed in Table 2. The
Nanodapter was configured in a direct
injection mode. A 75 µm × 25 cm C18column was kept at 60 °C and used for
peptide separation with a 90-minutegradient in a total 120-minute LC
run time.2,6For peptide identification, 5 μL
reconstituted MHC-I peptide samples
were injected once for each sample.
DDA was used with a selection of top
20 precursor ions. The detailed settings
for the 6550 iFunnel Q-TOF LC/MS arelisted in Table 3. For MHC-I peptide
analysis, singly charged ions wereselected as precursor ions even though
they were given lower priorities thandoubly and triply charged ions. Basedon literature, many MHC-I peptides
form singly charged ions, which should
be considered in DDA experiments.
However, higher collision energy should
be applied on singly charged ions thanmultiply charged ions.2

nanoESI source

Nanodapter

Agilent 1290 Infinity II UHPLC
Agilent 6550 iFunnel Q-TOF

Figure 3. Agilent Infinity UHPLC Nanodapter converts standard-flow LC to nanoflow LC coupled with anAgilent 6550 iFunnel Q-TOF LC/MS.
Table 2. Nano-LC parameters.
LC Conditions
Trap Column

PepMap C18, 75 µm × 2 cm, at 60 °C

Analytical Column

PepMap C18, 75 µm × 25 cm, at 60 °C

Solvent A

0.1% formic acid in water

Solvent B

0.1% formic acid in 90% acetonitrile

Flow Rate

0.085 mL/min primary flow
300 nL/min on-column flow rate

GradientTime 		(min)
%B
0390
3595
70

Injection Volume5 µLTime(min)97100120%B70
335Qualitative and quantitative analysis
using Byos workflow
Data analysis was processed using
Byos workflow from Protein Metrics Inc.
The Byos workflow includes peptide
identification using Byonic search engine
and peptide quantitation using Byologic
software (Figure 4). Byos workflow
processes peptide identification andquantitation consecutively with theparameters preset by users. Table 4lists some of the key parameters usedin Byonic and Byologic software for
MHC-I peptide analysis. Byonic was used
to search against the Uniprot humanprotein database with nonenzyme
specific and no limitation on missedcleavage. Oxidation (M, W) anddeamidation (N, Q) were set as variablemodifications. A manual score cut, 150,
was used for filtering identified peptides(Table 4A). The MS profiles for theidentified peptides were then extracted
in Byologic with certain retention times
and m/z windows. A decoy number of 50
was used as a second filter for protein
and peptide identification (Table 4B). The
unique peptides were summarized andquantified in Byologic.

Table 3. Agilent 6550 iFunnel Q-TOF LC/MS parameters.
ParameterValue

Spray Needle

New objective noncoated needle
25 µm id, 10 µm tip id, 5 cm lengthorthogonally positioned

Gas Temperature

200 °C

Drying Gas

11 L/minAcquisition Mode

Extended Dynamic Range (2 GHz)m/z 100 to 1,700
High analytical sensitivity

MSMS/MS

Mass Range

m/z 300 to 1,700

m/z 50 to 1,700

Acquisition Rate

3 spectra/sec>3 spectra/sec

Isolation Width

Medium (~m/z 4)

Collision Energy(Slope)*(m/z)/100+Offset
ChargeSlopeOffset
2
3.1
1
3
3.6-4.8
146>3
3.6-4.8

Max Precursor/Cycle

20Precursor Threshold

1,000 counts and 0.01%

Active Exclusion

Excluded after 1 spectra
Released after 0.2 min

Isotope ModePeptides

Sort Precursors

By charge state then abundance; +2, +3, 1, >+3

Scan Speed Varied Based On
Precursor Abundance

Yes

Target25,000 counts/spectrum

Use MS/MS Accumulation
Time Limit

Yes

Purity Stringency

100%

Purity Cutoff

30%

Table 4A. Byonic search parameters.

Table 4B. Byologic quantitation parameters.

Byonic Settings

Byonic

Peptide IDs

Byos

Quantitation

ByologicFigure 4. Byos workflow for peptide analysis.

6

Byologic Settings

Database

Uniprot human proteins

Enable In-Silico

NoDigestion Specificity

Nonspecific

Relevant Peptides

All types

Cleavage SideC-terminal

m/z Window

20 ppmApex Search Window

2 minutes

XIC Area Window

2 minutes

Enable Lock-Mass Calibration

No

Centroid Smoothing Width

0.02

Decoys50

Maximum Missed
Cleavage

No limit

Modifications

Oxidation (M, W)
Deamidated (N, Q)

Fragmentation TypeQTOF/HCD

Mass Tolerance

MS120 ppm

Manual Score Cut

150

MS240 ppm

Results and discussion

Sample 14,0000Sample 2×108Counts

Figure 5 is the total ion chromatogram(TIC) of the three samples using a
90-minute gradient with a total 2-hournano-LC run time. The TIC shows
reproducible retention time and peakabundance between the samples. Thetandem MS data were analyzed by
Byos workflow and peptide IDs weresummarized in Figure 6. The unique
MHC class I peptides identified in eachsample ranged from 2,284 to 2,426 witha CV% at 3.0%. The number of uniquepeptides identified across all threesamples is 3,604. A Venn diagram showsa more detailed relation of peptide IDs
between the samples. The number ofunique peptides identified in all threesamples is about 1,351, constituting
about 55.7% to 59.2% in each sample.
The same peptides identified between
two samples ranged from 65.4% to71.3% for each sample (Figure 7).

110Sample 3
×108
1Counts

MHC peptide identification andquantitation

Counts

×108

02040

60

80

100Acquisition time (min)Figure 5. TIC of MHC-I peptides using a 90-minute gradient.

Unique peptide number

3,500

Peptide ID number

3,000

Sample 2
2284

2,500

Sample 32355

277410491

2,0003604
1351

1,5001,0002426

2462355

2284

236

500
5930132Total

SampleFigure 6. The unique peptide number identified using an Agilent 6550 iFunnel Q-TOF LC/MS.

Sample 1
2426

Figure 7. Venn diagram of unique peptides
identified from the three samples.

7

Byologic extracts peptide abundance
based on the MS1 scan for eachidentified peptide. By adding the peptide
abundance, the total peptide abundancesin each sample are summarized inFigure 8 with a CV% at 11.1%. The
three samples were prepared using thesame six crosslinked PAW cartridges
on three different days. Multiple steps
were used to prepare the samples,
including crosslinking of mAb withPAW, GRANTA lysate loading on eachcartridge, combining the eluate, and asubsequent C18 cleanup. Therefore,
the CV% of the total peptide abundance
between the samples showed a good
day-to-day reproducibility using theautomated sample preparation. It also
demonstrated that the crosslinked
cartridges can be re-used and maintain agood performance.

Peptide length distribution

MHC peptide-binding motif analysis

The frequency distribution of peptide
length is plotted in Figure 9 to further
confirm the identification of MHC
class I peptides. Data shown in Figure 9
are compiled from the overall peptide
identified in Byos workflow analysis. The
3,604 unique peptides (Figures 6 and 7)
spanned peptide lengths from 3 to17 residues. However, the vast majorityof peptides (94%) were 8 to 11 residues
long, with most (75%) at 9 residues.
This is well in line with what has beenreported in literature.2

It is critical to carefully evaluate theMHC peptide-binding motif to ensurethe quality of the results. One popularmethod is to visualize positionsof residue preference within theimmunopeptidomic datasets. This canbe achieved using online tools suchas Seq2Logo: http://www.cbs.dtu.
dk/biotools/Seq2Logo/.
Since the majority of the peptides
identified were 8-, 9-, 10-, and 11-mers,
with 9-mers being the most abundant
peptides (>75%), all 9-mer sequenceswere uploaded to the Seq2Logo website
and generated the HLA peptide-binding
motif in Figure 10. The analysis of theHLA motif showed a strong preferencefor L or V at position 9 (C terminus) andat position 2.2

Peptide Abundance
1.2E+101E+10Counts

8E+09

6E+09

4E+09

2E+09

012Sample

Figure 8. MHC-I peptide abundance for each sample with CV% at approximately 11.1%.

8

3

80.00%75.30%

Frequency distribution of peptide length70.00%

60.00%% Peptide

50.00%

40.00%

30.00%

20.00%

9.98%
10.00%

5.26%
1.63%

0.00%

3

0.64%

0.23%

4

52.16%

3.48%

0.54%

678

910

110.49%

0.23%

0.05%

0.01%

0.00%

0.01%

121314151617Peptide length

Figure 9. The frequency distribution of the peptide length of MHC class I peptides from the average of three samples.

0.80.6Bits

0.4

0.20123456789Figure 10. MHC peptide-binding motif was constructed on the basis of nonamer peptides(created by Seq2Logo).

9Conclusion

References

An automated MHC-I associated peptide
enrichment using large-capacity (25 µL)
PAW cartridges for immunopeptidomics
analysis has been developed. This
workflow provides a reproducible
and easy-to-use enrichment for MHCpeptide analysis.

1.

Hunt, D. F. et al. Characterizationof Peptides Bound to the Class I
MHC Molecule HLA-A2.1 by Mass
Spectrometry. Science 1992,255(5049), 1261–1263.

2.

Purcell, A. W.; Ramarathinam,
S. H.; Ternette, N. Mass
Spectrometry-Based Identificationof MHC-Bound Peptides for
Immunopeptidomics. Nature
Protocols 2019, 14, 1687–1707.

3.

Caron, E. et al. Analysis of Major
Histocompatibility Complex (MHC)
Immunopeptidomes Using Mass
Spectrometry. Mol. Cell Proteomics
2015, 14(12), 3105–3117.

Antibody crosslinking with PAW
cartridge, immunoaffinity purification,and peptide cleanup can all beautomated on the AssayMAP Bravo in a
stepwise manner. AssayMAP 25 µL PAW
cartridges are well suited for processing
low-concentration MHC complex with ahigh loading speed and high enrichment
efficiency. 5 µL C18 cartridges provide
an efficient peptide separation andcleanup from protein complexes.
The unique MHC-I peptides identified
from the three samples prepared on
different days ranged from 2,284 to2,426 with a CV% less than 3%, whichshowed a highly consistent peptide IDresult. The peptide quantitation result
showed a good reproducibility of peptide
abundance from the three samples, witha CV% at about 11.1%. The day-to-day
sample comparison demonstrated agood reproducibility with an automatedworkflow, even with multiple sample
preparation steps. The peptide length
distribution from all the unique peptides
showed over 94% peptides identified
have lengths from 8 to 11 residues,
which is well in line with the literature.
The HLA peptide-binding motif generated
from the 9-mer sequence logo further
confirmed the preference of L and V atposition 9 and position 2.

www.agilent.com/chem
For Research Use Only. Not for use in diagnostic procedures.
DE.5387962963
RA.4412615741
This information is subject to change without notice.
© Agilent Technologies, Inc. 2020
Printed in the USA, September 18, 2020
5994-2344EN

4.

Wu, S.; Wu, L. F. Human
Breast Cancer Cell Line
Phosphoproteome Revealed by an
Automated and Highly Selective
Enrichment Workflow, Agilent
Technologies application note,publication number 5994-0315EN,
2018.