Comparison of High-Abundance Protein Depletion Techniques

for Biomarker Discovery

Life Science Group

2000 Alfred Nobel Drive
Hercules, CA 94547 USA

Tim Wehr,1 Chengjun Sun,1 Lei Li,1 Katrina Academia,1 Steve Freeby,1 Ning Liu,1 John Walker,1 Aran Paulus,1 and Chris Sutton2
1 Bio-Rad Laboratories, Hercules, CA 94547, USA; 2 Institute of Cancer Therapeutics, University of Bradford, Bradford, West Yorkshire BD7 1DP, UK

Introduction

Nano-LC-MALDI Workflow

To detect candidate biomarkers present in human plasma at

moderate-to-low abundance, high-abundance proteins (HAPs)
must be removed first. Several methods have been described
for HAP removal, including antibody affinity, peptide affinity,
and ion exchange. The goal of this study was to compare
an antibody-based affinity method with a peptide-based
affinity method for HAP removal from human plasma. Two
proteomic workflows were used. The first workflow, called
GeLC-MS, employed separation of HAP-depleted eluates
on a one-dimensional PAGE gel, followed by in-gel digestion
of lane segments and reverse-phase liquid chromatography
electrospray ionization tandem mass spectrometry (LCESI-MS-MS). The second workflow, LC-MALDI, employed
tryptic digestion of HAP-depleted eluates followed by nanoreverse- phase HPLC and matrix-assisted laser desorption/
ionization time-of-flight (MALDI-TOF-TOF-MS) analysis of
chromatographic fractions.

Apply 200 l plasma to

20 l ProteoMiner column

Apply 15 x 8 l plasma to
MARS-14 column

Elute with urea/CHAPS/HAc

Flowthrough

Precipitate protein and

reconstitute in
8 M urea + 2% CHAPS

Reduce and alkylate

Precipitate protein and

reconstitute
in 8 M urea + 2% CHAPS

Reduce and alkylate

Digest in situ with trypsin

Reduce and alkylate

The GeLC-MS workflow generated approximately 40 sets

of gel plugs for each sample, which required lengthy and
laborious in-gel digestion. The advantage of the LC-MALDIMS workflow was the ability to automate the entire process.
In the course of the investigation, an alternative strategy was
evaluated in which proteins bound to the peptide affinity
column were digested in situ with trypsin, followed by elution
and nano-LC-MALDI-MS analysis.

Digest with trypsin

Digest with trypsin

Fig. 2. Depletion of high-abundance human plasma proteins with ProteoMiner

column and MARS-14 column using the nano-LC-MALDI workflow.

GeLC-MS

30

55

ProteoMiner column
on-bead digestion

MARS-14 column
94 total

Nano-LC-MALDI

86 total
12
7

HPLC Conditions
Instrument

Flow rate

1100 Capillary LC
(Agilent Technologies)

UltiMate Intelligent LC Series

3000 (Dionex Corporation)

Magic C18 AQ, 0.2 x 50 mm

(Michrom Bioresources)

PepMap C18 0.075 x 150 mm

(Waters Associates)

3 l/min

300 nl/min

42

41

26

4
Gradient

550% ACN + 0.1% formic acid

1040% ACN + 0.1 formic acid
in 50 min

Fraction
collection

336 fractions codeposited

with matrix (a-cyano-4hydroxycinnamic acid) on an
AnchorChip MALDI plate
using a PROTEINEER fraction
collector (Bruker Daltonics)
MS Conditions

Search engine

LTQ linear ion trap

(Thermo Fisher) with ADVANCE
ESI source (Michrom
Bioresources)

ultraflex II MALDI-TOF/TOF
using WarpLC v.1.1 software
(Bruker Daltonics)

400 1600 m/z precursor scan,

MS-MS scans on top 5 most
intense ions

MS data used to compile match

list of all unique components
for MS-MS analysis

SEQUEST operating in
BioWorks 3.2 (Thermo Fisher)

11

ProteoMiner bead
solution digestion
82 total
Fig. 6. LC-MALDI identification of proteins from MARS-14 column flowthrough,
ProteoMiner on-bead digestion, and ProteoMiner solution digestion.

Conclusions
n

Bioinformatics
Mascot v.2.2
(Matrix Science)

Digest with trypsin

85 total

Fig. 5. Comparison of GeLC-MS and LC-MALDI for identification of human

plasma proteins depleted using ProteoMiner technology.

Analysis Conditions for GeLC-MS and Nano-LC-MALDI

Precipitate protein and reconstitute

in 8 M urea + 2% CHAPS

Reduce and alkylate

81 total

Search Swiss-Prot database using Mascot search and identify proteins

Scan conditons

Cut spots from 40 contiguous sections, 6 plugs/section

LC-MALDI-TOF-MS

Analyze by MALDI-TOF-TOF-MS

Flow through

Separate and stain with Coomassie Brilliant Blue R-250 stain (Bio-Rad)

47

GeLC-MS

51

Instrument

Load 2 x 25 g to Criterion Tris-HCl 8-16% linear gradient gel (Bio-Rad)

34

Spot 384 fractions onto MALDI target

Apply 15 x 8 l plasma to
MARS-14 column

Elute with urea/CHAPS/HAc

81 proteins total
47 unique proteins

Separate using nano-LC

GeLC-MS Workflow
Apply 200 l plasma to
20 l ProteoMiner column

78 proteins total
44 unique proteins

Fig. 4. Comparison of human plasma protein depletion techniques using

GeLC-MS.

Column

Results

ProteoMiner Column

44

Methodology
For antibody-based depletion, plasma was depleted of the top
14 most-abundant HAPs using a MARS-14 column (Agilent
Technologies). Proteins in the eluate were precipitated with TCA
and reconstituted in urea + CHAPS. For peptide-based depletion,
plasma was bound to a spin column packed with ProteoMiner
protein enrichment beads (Bio-Rad Laboratories), and proteins
were eluted with urea + CHAPS + acetic acid. For the GeLC-MS
workflow, the ProteoMiner column eluate or the MARS-14 column
flowthrough was separated on a 1-D gel, gel plugs were digested
with trypsin, and digests were analyzed by LC-MS-MS (Figures
1 and 3). For the nano-LC-MALDI workflow, the reconstituted
flowthrough from the MARS-14 column was digested with trypsin,
and the digest was separated by nano-HPLC. Fractions were
collected and analyzed by MALDI-MS-MS (Figures 2 and 3). For
proteins bound to the ProteoMiner beads, two approaches were
taken. In the first approach, proteins were eluted from the column
using urea + CHAPS + acetic acid. Proteins were precipitated
with TCA, reconstituted in urea + CHAPS, then digested with
trypsin and analyzed by nano-LC-MALDI-MS-MS. In the second
approach, proteins bound to the ProteoMiner beads were digested
in situ with trypsin, and the digest was analyzed by nano-LCMALDI-MS-MS (Figures 4, 5, and 6).

MARS-14 Column

Database

NCBI human

Swiss-Prot v.52.4 human

Fig. 3. Comparison of conditions used for GeLC-MS and nano-LC-MALDI

workflows.

 eLC-MS yields similar numbers of proteins for ProteoMiner

G
column- and MARS column-depleted plasma
 roteins identified by GeLC-MS in ProteoMiner column- and
P
MARS column-depleted plasma exhibit ~42% overlap
 eLC-MS and LC-MALDI identified similar numbers of proteins
G
in ProteoMiner column-depleted plasma
 f the total proteins identified in ProteoMiner columnO
depleted plasma by GeLC-MS and LC-MALDI, ~11% were
antibody sequences
 omparing depletion using MARS column, ProteoMiner
C
column with on-bead digestion, and ProteoMiner column using
solution digestion and LC-MALDI analysis:

MARS column is designed to work with a small (10 l) serum/

plasma sample volume; not ideal for 1D-MS-MS workflow
ProteoMiner columns are designed to work with a larger
(200 l) serum/plasma sample volume, producing more
protein than necessary for nano-LC-MALDI workflow
Similar numbers of proteins were identified for all
three techniques
Methods are complementary to each other

Analyze by LC-MS-MS

Search NCBI database and identify protein using SEQUEST program

Fig. 1. Depletion of high-abundance human plasma proteins with ProteoMiner
columns and MARS-14 columns using the GeLC-MS workflow.

Coomassie is a trademark of BASF Aktiengesellschaft. LTQ is a trademark of Thermo

Fisher Scientific Inc. PROTEINEER and AnchorChip are registered trademarks of Bruker
Daltonics GmbH Corporation. SEQUEST is a registered trademark of University of
Washington. UltiMate is a trademark of Dionex Corporation. ultraflex II is a trademark of
Bruker Corporation.ADVANCE is a trademark of Michrom Bioresources, Inc. BioWorks
is a trademark of Thermo Electron Corporation. Mascot is a trademark of Matrix
Science Ltd.
09-0293 Rev A
0309