A Proteomic Strategy for Increasing
Membrane Protein Identifications with
Use of the Agilent High Recovery mRP-C18
Reversed-Phase HPLC Column –A Comprehensive Protein Survey of the
HeLa Membrane Proteome
Application
Proteomics

Authors
James Martosella, Nina Zolotarjova, and Hongbin Liu
Agilent Technologies
2850 Centreville Road
Wilmington, DE 19808-1610
USA

Abstract
Membrane proteins play pivotal roles in various physiological processes such as signal transduction, moleculartransport, and cell-cell interactions and a comprehensive
analysis of these proteins is essential to uncovering diagnostic disease biomarkers, therapeutic agents and drug
receptor candidates. However, profiling membrane proteins has proven to be particularly challenging because of
their hydrophobic nature and low abundance. Theseobstacles pose major limitations for proteomic techniquessuch as gel electrophoresis or chromatography-based
separation methods. For electrophoretic analyses, manyhydrophobic proteins are not readily soluble causing poor
gel performance and recoveries, while liquid chromatography (LC) separation techniques may suffer from poor
separation characteristics, non-reproducibility and low
protein recoveries. To overcome these limitations, newproteomic technologies and strategies are constantly
under development. Here, we present a novel and highlyrobust method for the separation and identification of
HeLa cell membrane proteins by an LC-only based separation strategy. Employing optimized reversed phase (RP)conditions and using a uniquely designed reversed-phasecolumn material specifically engineered to enable highprotein recoveries, we have identified more than954 proteins (470 membrane and 337 integral membraneproteins) by HPLC-Chip LC/MS/MS. The optimized
reversed-phase (RP) separation and fractionation protocol for intact proteins, combined with TFE in-solution
digestion, represents a fast, reliable and reproducible tool
for the proteomic characterization of complex hydrophobic protein samples. We have demonstrated that thismethodology is a robust alternative to traditional
1D SDS-PAGE after RP fractionation, with the latter
requiring much more time and yielding fewer protein
identifications.

Introduction
Some of the most important cellular functions are
intrinsically tied to biological membranes and acomprehensive analysis of membrane proteins isessential for an in-depth understanding to uncoverdiagnostic disease biomarkers, therapeutic agentsand drug receptor candidates. Membrane proteinsplay pivitol roles in various physiologicalprocesses such as signal transduction, cell-cell contact, the selective transport of molecules and otheressential functions. The significance of membraneproteins in drug discovery and drug development
is evidenced by the fact that about two-thirds of all
drug targets are directed towards these proteins.
However, profiling membrane proteins has provento be particularly challenging because of their low
abundance and the difficulties in resolving andidentifying them due to their hydrophobic nature.

Traditionally, proteomic analyses of complex protein samples involve the resolution of proteinsusing 1D or 2D gel electrophoresis (GE) followedby the identification of resolved proteins by mass
spectrometry or simply by shotgun proteomics
methods. However, the limitations of the electrophoretic separation, such as protein size,
extreme pI range and proteins insolubility limit
the ability of these methods. Limited dynamic
range of detection is also an issue because membrane proteins are typically lower in abundance
when compared with soluble proteins. As an alternative approach, reversed-phase high-performance
chromatography has been used for resolving membrane proteins and peptides and is used as ameans to reduce sample complexity, perhaps prior
to GE. But, chromatography of high-molecularmass and hydrophobic proteins also presents its
own challenges that often prohibit its use. Column
chromatography of hydrophobic proteins can present sample-specific obstacles for researchers andoften requires specific expertise in sample solubilization techniques, method development andknowledge of column material types. In addition,
RP separations of complex protein samples havesuffered from low sample recoveries, poor reproducibility and inadequate resolution. Protein
recoveries from RP chromatography typicallyrange from 30%–75% and most column materials
do not provide the resolution needed for highly
complex sample mixtures, such as those presented
by membrane proteins.

prior to LC/MS/MS analysis. The optimized RP
column prefractionation workflow saved considerable sample preparation time and labor, by allowing the omission of an additional SDS-PAGE
preparation step, and enabled the identification of
more proteins. To date, we have identified 954 proteins (470 membrane and 337 integral membrane)
associated with a HeLa membrane sub-fraction.
Among the protein identifications are many
important cell receptors, identified in Table 1.

HeLa Cell Membrane Proteins

Solubilization

RP-HPLC

Strategy 1
In-Solution Digestion
mAU
8
7
6
5
4
3
2
1
0
0

In this study, two proteomic sample preparation
strategies were evaluated for enabling a comprehensive survey of membrane protein identifications and method robustness with carefulconsideration given to time consumption andhands-on labor. In the first method (gel-free),
HPLC fractions were tryptically digested in-solution and analyzed by 2D HPLC-Chip LC/MS/MS. In
the second method (gel-based), the HPLC fractions
were further resolved on SDS-PAGE and all gelbands from 9 lanes excised (216 bands in total),
tryptically digested, and analyzed by 1D HPLCChip LC/MS/MS (Figure 1). Thus, we found that 1D
prefractionation alone, with the Agilent mRP-C18
column, sufficiently reduces sample complexity

2

10

20

30

2D nano-chip
LC/MS/MS

Figure 1.

40

50

60

Strategy 1
In-Gel Digestion70

80 min

mAU
8
7
6
5
4
3
2
1
0
0

10

20

30

40

50

60

70

1D nano-chip
LC/MS/MS

Sample workflows for the identification of HeLa
membrane proteins isolated from HeLa cells.

80 minTable 1.

Cell Receptor Proteins Iidentified by HPLC-Chip LC/MS/MS Using the In-solution Digestion Strategy Shown in Figure 1.

entry_name
28 kDa Golgi SNARE protein (Golgi SNAP receptor complex member 1) (28 kDa cis-Golgi
SNARE p28) (GOS-28)
40S ribosomal protein SA (p40) (34/67 kDa laminin receptor) (Colon carcinoma lamininbinding protein) (NEM/1CHD4) (Multidrug resistance-associated protein MGr1-Ag)
Atrial natriuretic peptide receptor A precursor (ANP-A) (ANPRA) (GC-A) (Guanylate cyclase)(EC 4.6.1.2) (NPR-A) (Atrial natriuretic peptide A-type receptor)
Autocrine motility factor receptor, isoform 2 (EC 6.3.2.-) (AMF receptor) (gp78)
B-cell receptor-associated protein 31 (BCR-associated protein Bap31) (p28 Bap31) (CDM
protein) (6C6-AG tumor-associated antigen) (DXS1357E)
cAMP-dependent protein kinase type II-alpha regulatory subunit
Cation-dependent mannose-6-phosphate receptor precursor (CD Man-6-P receptor) (CDMPR) (46 kDa mannose 6-phosphate receptor) (MPR 46)
Cation-independent mannose-6-phosphate receptor precursor (CI Man-6-P receptor) (CIMPR) (M6PR) (Insulin-like growth factor II receptor) (300 kDa mannose 6-phosphate
receptor) (MPR 300) (MPR300) (CD222 antigen)
CD44 antigen precursor (Phagocytic glycoprotein I) (PGP-1) (HUTCH-I) (Extracellular matrix
receptor-III) (ECMR-III) (GP90 lymphocyte homing/adhesion receptor) (Hermes antigen)(Hyaluronate receptor) (Heparan sulfate proteoglycan) (Epi
CD97 antigen precursor (Leukocyte antigen CD97)
Coxsackievirus and adenovirus receptor precursor (Coxsackievirus B-adenovirus receptor)(hCAR) (CVB3 binding protein)
Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 63 kDa subunit precursor(EC 2.4.1.119) (Ribophorin II) (RPN-II) (RIBIIR)
Ephrin type-A receptor 2 precursor (EC 2.7.1.112) (Tyrosine-protein kinase receptor ECK)
(Epithelial cell kinase)
Epidermal growth factor receptor precursor (EC 2.7.1.112) (Receptor tyrosine-protein kinase ErbB-1)
Folate receptor alpha precursor (FR-alpha) (Folate receptor 1) (Folate receptor, adult) (Adult
folate-binding protein) (FBP) (Ovarian tumor-associated antigen MOv18) (KB cells FBP)
G-protein coupled receptor family C group 5 member C precursor (Retinoic acid induced
gene 3 protein) (RAIG-3)
Inositol 1,4,5-trisphosphate receptor type 1 (Type 1 inositol 1,4,5-trisphosphate receptor)(Type 1 InsP3 receptor) (IP3 receptor isoform 1) (InsP3R1) (IP3R)
Inositol 1,4,5-trisphosphate receptor type 3 (Type 3 inositol 1,4,5-trisphosphate receptor)(Type 3 InsP3 receptor) (IP3 receptor isoform 3) (InsP3R3)
Integrin alpha-2 precursor (Platelet membrane glycoprotein Ia) (GPIa) (Collagen receptor)(VLA-2 alpha chain) (CD49b)
Integrin alpha-3 precursor (Galactoprotein B3) (GAPB3) (VLA-3 alpha chain) (CD49c) (FRP-2)
Integrin alpha-5 precursor (Fibronectin receptor alpha subunit) (Integrin alpha-F) (VLA-5) (CD49e)
Integrin alpha-V precursor (Vitronectin receptor alpha subunit) (CD51 antigen)Integrin beta-1 precursor (Fibronectin receptor beta subunit) (CD29 antigen) (Integrin VLA-4 beta subunit)
Integrin beta-4 precursor (GP150) (CD104 antigen)Integrin beta-5 precursor
Keratin, type II cytoskeletal 1 (Cytokeratin 1) (K1) (CK 1) (67 kDa cytokeratin) (Hair alpha protein)
Lamin B receptor (Integral nuclear envelope inner membrane protein) (LMN2R)
Lysosome membrane protein II (LIMP II) (Scavenger receptor class B, member 2) (85 kDalysosomal membrane sialoglycoprotein) (LGP85) (CD36 antigen-like 2)
Membrane associated progesterone receptor component 1 (mPR)
Microsomal signal peptidase 23 kDa subunit (EC 3.4.-.-) (SPase 22 kDa subunit) (SPC22/23)(UNQ1841/PRO3567)
Mitochondrial import receptor subunit TOM22 homolog (Translocase of outer membrane 22
kDa subunit homolog) (hTom22) (1C9-2)

accession_number numPepsUnique scoreUnique protein_mw

Q9NS69Mitochondrial precursor proteins import receptor (Translocase of outer membrane TOM70)
Neogenin precursor
Ninein (hNinein)
Orphan nuclear receptor TR4 (Orphan nuclear receptor TAK1)
Plexin B2 precursor (MM1)
Polymeric-immunoglobulin receptor precursor (Poly-Ig receptor) (PIGR) [Contains: Secretory component]
Receptor-type tyrosine-protein phosphatase F precursor (EC 3.1.3.48) (LAR protein)(Leukocyte antigen related)
Receptor-type tyrosine-protein phosphatase S precursor (EC 3.1.3.48) (R-PTP-S) (Proteintyrosine phosphatase sigma) (R-PTP-sigma)
Selenoprotein S (VCP-interacting membrane protein) (AD-015) (SBBI8)Signal recognition particle receptor alpha subunit (SR-alpha) (Docking protein alpha) (DP-alpha)Signal recognition particle receptor beta subunit (SR-beta) (Protein APMCF1)

O94826
Q92859Q9P2E9P49116
O15031
P01833
P10586
Q13332Q9BQE4
P08240
Q9Y5M8

O952492

21.2828612.8

P08865

4

56.96

32723

P16066Q9UKV5

2
2

21.37
27.85

118919.972996.2

P51572
P13861

6
2

81.03
27.07

27860.645387.4

P20645345.8430993.5

P11717

659.58

274277.4

P16070
P4896048

58.84
105.8181554
91841.9

P783108

108.9840030.1

P04844

6

108.4369284.3

P29317
P0053338

33.06
115.99108254.9134278.3

P153289130.4129819.3

Q9NQ842

21.1948193.5

Q14643566.26

313946.8

Q14573679.03304040.1

P17301P26006
P08648
P06756
P05556
P16144
P18084
P04264
Q14739

5
4
4
7
19
12
3
52
8

78.71
41.21
55.25
91.39
235.8
176.15
41.86
849.2
89.47

129296.1
118698.4
114537.1
116052.5
88466
202152.3
88054.965886.870703.6

Q14108O002642
11

26.57
157.7454159.321540.1

P61009674.61

20313.52

36.4615521.7

13
2
23
2
23
5

200.1125.44
327.7220
299.0577.4967455.2
159960.3
152472.965414.9205100.3
83314

3

32.21

211845.8

3
3
12
13

32.81
47.63
147.98
192.78217095.521116.2
69811.6
29702.4

3

Experimental
The high-recovery macroporous reversed-phase
C18 column (mRP-C18) for separating proteins [1]is a product from Agilent Technologies (Wilmington, DE). A 4.6-mm × 50-mm mRP-C18 column(part number 5188-5231) was used with an automated Agilent 1100 LC system with a thermostatted autosampler equipped with a 900 µL injection
loop, quaternary pump, thermostatted analyticalscale fraction collector and column heating at
80 °C. The reversed-phase separations of HeLa
membrane proteins were performed under a set of
optimized conditions using a quaternary mobile
phase system consisting of multi-segmented andlinear elution gradient, with eluent A (0.1% TFA inwater, [v/v]), eluent B (0.08% TFA in acetonitrile,[v/v]), eluent C (20% formic acid in acetonitrile[v/v]) and eluent D (2-propanol). The gradient flowrate was 0.75 mL/min and detection was monitored at 280 nm. For consecutive chromatographic
runs, a 30-minute post-run comprised of 20.0%
eluent B was added to reequilibrate the column.

formic acid and briefly sonicated for 30 seconds ina water bath. The samples were then re-dried in
the centrifugal vacuum concentrator, resolubilized
in 500 µL of 80% formic acid and again briefly sonicated for 30 seconds in a water bath. The final
sample concentration was approximately 0.58 µg/µL
in 80% formic acid. HPLC injection amounts varied
from 200 µL to 500 µL depending on the amount ofprotein needed for either in-solution digestion or
SDS PAGE analysis.
After formic acid solubilization, the samples wereseparated under high temperature RP conditions
using a combination of a multisegmented elution
gradient of water (0.1% TFA)/ACN (0.08% TFA) anda linear elution gradient of ACN (20% formicacid)/2-propanol (Table 2). HPLC fraction collection was performed by time, collecting 1.5 minutetime slices starting at 1.0 minute and continuing to70.0 minutes. The fractions were collected into
1.5-mL plastic tubes (part number 5188-5251) at
4 °C. The fractions were then dried in a centrifugal
vacuum concentrator (Thermo-Savant, Millford,MA) and stored at –80 °C.

HeLa Membrane Sample Preparation and Solubilizationfor HPLC Column Loading
Table 2.

Isolation of membranes from HeLa S3 cells was
performed by a modified carbonate fractionation
procedure [2, 3]. HeLa S3 cells were grown to 90%
confluency in Ham’s F12 medium with 2 mM
L-glutamine and 1.5 g/L sodium bicarbonate supplemented with 10% fetal bovine serum. Afterwashing with PBS, cells were collected and washed
one additional time with PBS and then with 10 mM
Tris-HCl, pH-7.0. Cells were centrifuged and thepellet was resuspended in the above Tris buffer
with “Complete” protease inhibitors. Cells wereallowed to swell for 10 min, and gently homogenized in a tight-fitting Dounce homogenizer(20 strokes). Unbroken cells and debris were
removed by centrifugation at 3200 × g for 10 minutes
and the cell lysate was diluted with ice-cold 100 mM
sodium carbonate, pH-11.5, to a final protein concentration of 0.28 mg/mL. After incubation at
4 °C for 1.5 hr. (slow rotation), lysate was centrifuged at 103,900 × g for 1 hr. at 4 °C. Membranepellets were rinsed gently with the ice cold waterand then with 10 mM Tris-HCl, pH-7.4. Membranes
were aliquoted and stored at –80 °C.
Prior to HPLC injection, the membrane fractions
required dissolution in formic acid to enable injection onto the column. As needed, 100 µL aliquots(292 µg) of the membrane fraction were dried in acentrifugal vacuum concentrator (Thermo-Savant,Millford, MA), resolubilized in 200 µL of 80%

4

Lipid Raft Fractionation Multisegmented Gradient

Flow
Stoptime
Posttime
Column temp.
Solvent A
Solvent B
Solvent C
Solvent D
Detection UV
Pressure limit

0.75 mL/min
86.0 min30.0 min
80.0 ºC
Water/0.1% TFA
ACN/0.08% TFA
ACN/20% Formic acid
2-Propanol
280 nm
250 barGradient
Time (min)
0
14
50
75
77
78
80
83
86

%B
20
34
50
100
100
0
0
0
20

%C
0
0
0
0
0
100
100
0
0

%D
0
0
0
0
0
0
0
100
0

Electrophoretic Analysis
SDS-PAGE analysis was performed on Invitrogen
Tris-glycine precast gels (4%–20% acrylamide,
10 wells, 1 mm). Fractions from mRP-C18 RP separations were combined based on UV absorbance at
280 nm and dried in a SpeedVac on low heat. Following resuspension in 2x sample preparationbuffer, fractions were heated for 1 min. at 50 °C,

and then loaded onto the gel. Gel-separated proteins were visualized by Coomassie Blue staining
using Pierce GelCode Blue (part number 24592).
In-Solution and In-Gel Digestion
In-gel digestion and HPLC-Chip-LC-MS/MS(Gel-Based Method)
Forty-seven mRP-C18 column fractions, collected
in 1.5 minute time slices from 0–70 minutes during
the separation shown in Figure 2, were combined(based on previous gel pattern results) and loaded
into 9 gel lanes. Twenty-four bands from each lane(216 in total) were excised and digested withtrypsin using an Agilent in-gel digestion kit (partnumber 5188-2745). The digested peptides wereextracted and proteins identified by LC-MS/MS
analysis on an Agilent 1100 HPLC-Chip coupled to
the XCT Ultra Ion Trap. The digests were loaded
onto a standard RP chip (Agilent, Zorbax 300SBC18, 5-µm, 0.075-mm id × 43-mm, part number
G4240-62001). Elution of peptide fragments was
accomplished by RP gradient elution with buffer
A; water/3.0% ACN (0.1% formic acid) and buffer
B; water/95% ACN (0.1% FA). The XCT Ultra ion
trap mass spectrometer was operated in standard
scan mode for MS analysis and in ultra scan modefor MS/MS.
The gradient for the RP separations is as follows:
Time (min)

0

2

20

22

22.5 23.5 24

%B

3

10

35

50

95

95

3

In-solution digestion and 2D HPLC-Chip-LCMS/MS (Gel-Free Method)
Forty-Seven mRP-C18 column fractions were combined into 17 fractions and each combined fraction
was dried and digested with trypsin using a TFE
digestion protocol [4]. The digested fractions wereanalyzed with an Agilent 1100 nano-twodimensional-LC and 1100 MSD trap XCT ultra. The
digests were first loaded onto a capillary SCX
column (0.25-mm id × 40-mm) and eluted with aseries of 2 µL ammonium acetate solution injection
with increased concentration.
SCX elution steps (mM):
1
20

2
50

3
4
5
6
7
8
9
10
11
100 150 200 250 350 500 700 1000 2000

The eluted peptides from each salt step were further separated with the same RP chip and gradientas above with the same MS operating conditions.
Total analysis time for each combined fraction was
about 6 hours.

MS/MS data were searched against the SwissProt
Human database (total of 12,015 entries), using
Spectrum Mill computer database search algorithm, with the “Calculate Reversed Database
Scores” option “on”. The peptide/protein hits were
filtered with the “autovalidation” option using thefollowing parameters: minimum score for peptides:+1, 7.0; +2, 8.0 (if SPI larger than 90%, the score
was lowered to 7.0); +3, 9.0; +4, 9.0. All peptide
matches required a “Forward-Reverse Score”
larger than 1.0, and “Rank 1–2 score” larger than
1.0. The protein score was set at a minimum of
15.0. Only fully tryptic peptides were considered,with two missed cleavages allowed.
All MS data from both in-gel and in solution
approaches were searched against SwissProt
Human database (total 12,015 entries), using
Spectrum Mill (Agilent) computer databasesearch algorithm.

Results and Discussion
The Agilent high-recovery macroporous reversedphase C18 column (mRP-C18) column was used toseparate HeLa membrane proteins isolated from a
HeLa cell total lysate. Using the Agilent mRP-C18
column and optimized RP chromatographic conditions we collected column fractions and performed
a comparative comprehensive analysis of protein
identifications by nano-chip LC/MS/MS for protein
fractions directly in-solution digested, from RP
HPLC, versus an identical set of fractions RP collected and further resolved by SDS-PAGE andin-gel digested.
To evaluate the utility of either strategy (gel-freeversus gel-based), an efficient chromatographic
separation was established for fraction processing
and analysis. Figure 2 is a representative RP chromatogram for the separation of HeLa membraneproteins on a 4.6-mm × 50-mm mRP-C18. Changes
to gradient compositions and elution times weresystematically performed to optimize this separation, while SDS-PAGE of collected fractions wasused to characterize the separation efficiency. The
elution conditions and column material enabled awell resolved protein separation and displayed
excellent peak shapes ideal for discrete fraction
collection. The area of the chromatogram from
0–70 minutes represents the region of highest protein elution. Within this region, we collected 47 RP
fractions by time-based autosampling and resolved
them on an SDS-PAGE (Figure 3). The electrophoretic analysis of the UV profile details thehighly separated proteins and the discrete protein
banding patterns.
5

mAU
4.5
4.0

Absorbance at 280 nm

3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0

Figure 2.

10

20

30

40
Time (min.)

50

60

70

80

Reversed-phase separation of 300 µg HeLa membrane sub-fraction from a 4.6-mm × 50-mm mRP-C18 column.
The region from 0–70 minutes represents the area of highest protein elution as determined by SDS PAGE
(shown in Figure 3).

1

2

3

4

5

6

7

8

9

10

1

2

3

4

5

6

7

8

9

10

kDa
200.0
116.3
97.4
66.3
55.4
36.5
31.0
21.5
14.4
6.0

Gel 1
1
2
3
4
5
6
7
8
9
10

Figure 3.

6

HeLa membranes, starting material, 22 µg
Mark12 standards
Fractions
1–4
Fractions
5–8
Fractions
9–12
Fractions
13–16
Fractions
17–20
Fractions
21–24
Fractions
25–28
Fractions
29–32

Gel 2
1
2
3
4
5
6
7
8
9
10

Mark12 standards
Fractions
Fractions
Fractions
Fractions
Fractions
Fractions
Fractions
Fractions
Fraction

33–36
37–40
41–44
45–48
49–52
53–56
57–60
61–64
65–70

SDS PAGE of a HeLa membrane sub-fraction prefracationated by an mRP-C18 chromatography column (see Figure 2).
Forty seven fractions were collected at 1.5 minute time intervals from 0-70 minutes and uniquely combined based on
previous SDS-PAGE.

Upon establishing an optimized separation protocol and after evaluating SDS PAGE analyses likethat shown in Figure 3, RP fractions were futherconsolidated and, combined for mass spectrometry
analysis by either direct in-solution digestion or by
SDS PAGE and gel band digestion. To compare protein identification results from both approaches,
MS data acquisition time was kept the same. For
the in-solution method, a total of 17 uniquely combined fractions were tryptically digested and analyzed by 2D HPLC-Chip- LC/MS/MS, with a total
MS time of approximately 105 hours. For the gel
based method, a total of 216 bands form 9 SDS
PAGE lanes were analyzed by 1D HPLC-ChipLC/MS/MS and resulted in a total of 108 hours MS
time.
MS results for the gel-free method showed a totalof 954 proteins identified with 470 being membrane proteins and 337 of those being integral
membrane proteins. Alternatively, results by thegel-based approach gave 688 total proteins with
364 membrane protein identifications and
286 integral membrane identifications. Thus, total
protein identifications from the in-solution method
were almost 40% greater than the latter method,while the membrane protein identifications were30% more. In our study, the time saved to omit the
SDS PAGE separation and analysis was more than
4 days. Furthermore, the SDS-PAGE method
required extensive sampling handling and manipulation effort that was not needed by direct
in-solution digestion.

Conclusion
In this study, we showed the advantage of performing mass spectrometric analysis directly following
HPLC fractionation (in-solution digestion) of intact membrane proteins without the need for subsequent protein separation by gel-based methods.
Employing an HPLC fractionation “only” strategy
and utilizing in-solution tryptic digestion and twodimensional separation of peptides, we identified
954 proteins (470 membrane and 337 integral
membrane) associated with a HeLa membrane subfraction. Among these membrane identifications
are important pharmaceutical targets, such as EGF
receptors, Integrin proteins, TNF receptors, etc.
(Table 1).

membrane proteome without the need for additional protein separation. The column has previously shown excellent reproducibility and highprotein recoveries for hydrophobic sample types,
such as lipid rafts [5]. The optimized separation
protocol is easy to follow and demonstrates excellent utility for use in proteomic workflows for separating, and fractionating, complex sample
mixtures such as membrane proteins.

Reference
1. J. Martosella, N Zolotarjova, H. Liu, G Nicol, andB. E. Boyes, “Reversed-phase High-performance
Liquid Chromatographic Prefractionation of
Immunodepleted Human Serum Proteins toEnhance Mass Spectrometry Identification ofLower-abundant Proteins”. (2005) J Proteome
Res, 4, (5), 1522-37.
2. J. Blonder, M. B. Goshe, R. J. Moore,
L. Pasa-Tolic, C. D. Masselon, M. S. Lipton and
R. D. Smith, “Enrichment of Integral Membrane
Proteins for Proteomic Analysis Using Liquid
Chromatography – Tandem Mass Spectrometry”.
(2002), Journal of Proteome Research, 1,
351–360.
3. Y. Fujiki, A. L. Hubbard, S. Fowler and
P. B. Lazarow, “Isolation of Intracellular Membranes by Means of Sodium Carbonate Treatment: Application to Endoplasmic Reticulum”.
(1982), The Journal of Cell Biology, 93, 97–102.
4. J. E. Meza, C. A. Miller, S. M. Fischer, “Improved
Tryptic Digestion of Proteins Using 2,2,2-Trifluoroethanol (TFE)”. Poster presentation at the
Association of Biomolecular Resource Facilities
Symposium in Portland, Oregon during
February 28 thru March 2, 2004.
5. J. Martosella, N. Zolotarjova, H Liu, S. C. Moyer,
P. A. Perkins, and B. E. Boyes, “High Recovery
HPLC Separation of Lipid Rafts for Membrane
Proteome Analysis”. (2005), J Proteome Res,
accepted.

For More Information
For more information on our productrs and services,
visit our Web sit at www.agilent.com/chem.

The Agilent high-recovery mRP-C18 column andoptimized chromatographic conditions provided ahigh degree of resolution and enabled sufficient
reduction in sample complexity to profile a HeLa

7

www.agilent.com/chem

Agilent shall not be liable for errors contained herein or for incidental or consequential
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Information, descriptions, and specifications in this publication are subject to change
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© Agilent Technologies, Inc. 2006
Printed in the USA
April 27, 2006
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