Research
Expression Profiling of Lymphocyte Plasma
Membrane Proteins*□
S
Matthew J. Peirce‡, Robin Wait, Shajna Begum, Jeremy Saklatvala,
and Andrew P. Cope
The physicochemical properties of plasma membrane
proteins of mammalian cells render them refractory to
systematic analysis by two-dimensional electrophoresis.
We have therefore used in vivo cell surface labeling with a
water-soluble biotinylation reagent, followed by cell lysis
and membrane purification, prior to affinity capture of
biotinylated proteins. Purified membrane proteins were
then separated by solution-phase isoelectric focusing and
SDS-PAGE and identified by high-pressure liquid chromatography electrospray/tandem mass spectrometry. Using
this approach, we identified 42 plasma membrane proteins from a murine T cell hybridoma and 46 from unfractionated primary murine splenocytes. These included
three unexpected proteins; nicastrin, osteoclast inhibitory
lectin, and a transmembrane domain-containing hypothetical protein of 11.4 kDa. Following stimulation of murine splenocytes with phorbol ester and calcium ionophore, we observed differences in expression of CD69,
major histocompatibility complex class II molecules, the
glucocorticoid-induced TNF receptor family-related gene
product, and surface immunoglobulin M and D that were
subsequently confirmed by Western blot or flow cytometric analysis. This approach offers a generic and powerful
strategy for investigating differential expression of surface proteins in many cell types under varying environmental and pathophysiological conditions. Molecular &
Cellular Proteomics 3:56 – 65, 2004.
The plasma membrane (PM)1 constitutes the interface between eukaryotic cells and their external environment. ConFrom the Kennedy Institute of Rheumatology Division, Faculty of
Medicine, Imperial College London, London W6 8LH, United Kingdom
Received, July 3, 2003, and in revised form, October 10, 2003
Published, MCP Papers in Press, October 21, 2003, DOI
10.1074/mcp.M300064-MCP200
1
The abbreviations used are: PM, plasma membrane; 2DE, twodimensional gel electrophoresis; MS/MS, tandem mass spectrometry; ICAT, isotope coded affinity tag; IEF, iso-electric focusing;
SA, streptavidin; sulfo-NHS-LC-biotin, sulphosuccinimidyl-6-(biotinamido) hexanoate; sulfo-NHS-SS-biotin, sulphosuccinimidyl-2-(biotinamido) ethyl-1,3 dithiopropionate; PNS, post-nuclear supernatant;
GRAVY, grand average of hydrophobicity; GRP78, glucose-regulated
protein of 78kDa; GITR, glucocorticoid-induced tumor necrosis factor
receptor-related; OCIL, osteoclast inhibitory lectin; HPLC, high-pressure liquid chromatography; PMA, phorbol 12-myristate 13-acetate;
PBS, phosphate-buffered saline; LAT, linker for activation of T cells;
1DE, one-dimensional gel electrophoresis; IPG, immobilized pH gra-
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Molecular & Cellular Proteomics 3.1
sequently, the functions of proteins embedded in this membrane include cell/cell and cell/extracellular matrix
recognition, the reception and transduction of extracellular
signals, and the transport of solutes and water molecules into
and out of the cell. Abnormal PM protein expression has
profound biological effects and may, for example, underlie
phenotypic and functional differences between normal and
tumor cells (1). For these reasons, profiling of PM protein
expression is an area of intense interest (2).
Although about 25% of open reading frames in fully sequenced genomes are estimated to encode integral membrane proteins (3), global analysis of membrane protein expression has proved problematic (4). Two-dimensional gel
electrophoresis (2DE) can resolve over a thousand proteins on
a single large format gel, but PM proteins are often severely
underrepresented in 2DE protein patterns because of their
large size, low abundance, and hydrophobicity (5). Although
the introduction of improved chaotropes such as thiourea (6)
and the development of novel zwitterionic detergents (6 – 8)
has improved matters, particularly for prokaryotic organisms,
the detection of integral membrane proteins from higher eukaryotes by 2DE remains unsatisfactory (9).
One strategy to increase the representation of membrane
proteins on two-dimensional gels would be to pre-enrich the
membrane proteins prior to electrophoresis. Several groups
have attempted this using the established technique of cell
surface biotinylation (10) and affinity capture with avidin (11–
13). However, because the approach does little to address the
intrinsic limitations of 2DE, it is unsurprising that the number
of membrane proteins characterized remains disappointing;
18 in a recent study of the gastric pathogen Helicobacter
pylori (11) and 14 in an investigation of human tumor cell lines
(13), which, in the latter study, were accompanied by a large
number of heat shock proteins, not normally thought of as PM
proteins.
An alternative approach is to omit the isoelectric focusing
(IEF) step and apply an enriched membrane protein fraction
directly to SDS-PAGE, thus overcoming the solubility and
transfer problems intrinsic to 2DE (14, 15). Because the high
resolution of 2DE is sacrificed, most bands on the gel contain
multiple comigrating proteins, but the ability of high-pressure
dient; TTBS, Tween/Tris-buffered saline; BSA, bovine serum albumin;
FACS, fluorescence-activated cell sorting; HRP, horseradish peroxidase; Ig, immunoglobulin; MHC, major histocompatibility complex.
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
This paper is available on line at http://www.mcponline.org
Protein-MS Analysis of Plasma Membrane Proteins
liquid chromatography (HPLC) tandem mass spectroscopy
(MS/MS) to analyze complex mixtures of peptides enables the
use of a conventional strategy of tryptic digestion and mass
spectrometric identification. A recent study using this approach identified over 500 proteins in breast cancer cell membranes, including several putative tumor-specific markers
(14).
“Shotgun” methods take this approach a stage further, and
proteolytically digest the sample without prior electrophoretic
separation. The resulting mixture of thousands of peptides are
then separated by sequential strong cation exchange and
reverse-phase chromatography before identification by HPLC
MS/MS (16, 17). This approach offers considerable advantages for membrane protein characterization because (provided efficient digestion can be effected) separation is performed at the peptide rather than protein level, so most of the
solubility problems associated with hydrophobic proteins are
obviated.
When used in conjunction with the isotope-coded affinity
tagging (ICAT) strategy (18), it is possible to determine quantitative protein expression profiles. Thus, Aebersold and colleagues identified 491 proteins in a microsomal membrane
fraction of HL-60 cells. Of these, 55 (11.2%) were known cell
surface antigens, receptors, and membrane proteins and 24
(4.9%) were channel proteins. Furthermore, the expression of
eight PM proteins differed more than 2-fold between phorbol
ester-treated cells (19).
The disadvantage of most shotgun strategies is that computationally intensive analysis of the entire dataset is always
required, even when only a few proteins display changes in
expression levels. Furthermore, no information on the charge
or apparent mass of the intact proteins is obtained, and all
connectivity between parent proteins and their peptide digestion products is lost, which hinders characterization of posttranslational modifications.
Multidimensional chromatography is therefore best regarded as a complement rather than a replacement for gelbased proteomics, because it appears that there are substantial subsets of cellular proteins that remain undetected unless
both methodologies are applied in tandem. For example, in a
recent comprehensive analysis of the proteome of Oryza sativa (rice), 47% of the leaf proteins identified from two-dimensional gels were not detected by multidimensional chromatography, while a substantial number of proteins identified by
the latter technique were not observed on the gels (20).
In the present report, we describe an improved cell-surface
biotinylation/streptavidin (SA) affinity capture methodology for
profiling membrane proteins. The resulting mixture of surfacederived proteins can in principle be analyzed either by shotgun or conventional methods. However, we adopted a gelbased approach because this technology is available to the
majority of the proteomics community and is more readily
integrated into biochemical purification strategies. We have
circumvented the major limitations of conventional 2DE by
adopting solution-phase IEF and one-dimensional gradient
SDS-PAGE prior to trypsinolysis and HPLC MS/MS of recovered proteins. With this approach, we have identified 74 PM
proteins from a T cell clone and primary murine splenocytes.
Of these, 12 appeared to be differentially expressed by cells
activated with phorbol ester and ionomycin. Six of these
putative activation-induced changes were selected for further
study and independently verified by immunoblotting or flow
cytometry. This technique should provide a useful complement to existing proteomic approaches for cataloguing and
comparing PM protein profiles in diverse cell types.
EXPERIMENTAL PROCEDURES
Cell Culture—The mouse T cell hybridoma clone 11A2 was derived
and propagated in RPMI 1640 containing 10 mM glutamine and 25 mM
HEPES (Biowhittaker, Wokingham, UK) further supplemented with:
10% fetal calf serum (Labtech, Ringmer, UK); 10 mM sodium pyruvate
(Sigma, Poole, UK); 10 U/ml penicillin/streptomycin (Biowhittaker),
and 10 mM ␤-mercaptoethanol (Life Technologies, Inc., Paisley, UK)
as previously described (21).
Cell Isolation—Splenocytes were derived from 8 –12-week-old
male or female DBA1/J H-2q mice. Single-cell suspensions were
prepared by standard techniques prior to culture at a density of 107
cells/ml in complete medium in the presence or absence of phorbol
12-myristate 13-acetate (PMA) (30 ng/ml) and ionomycin (30 ng/ml),
both purchased from Sigma.
Cell Surface Biotinylation—Cells were washed three times and
resuspended (2–5 ⫻ 107/ml) in BBS (10 mM boric acid, 2.3 mM sodium
tetraborate, 115 mM NaCl, pH 8.1) in which biotinylation of lysines
proceeds more efficiently than in phosphate-buffered saline (PBS)
(22). Cells were biotinylated (20 min, 4 °C) using 0.1 mg/ml sulfosuccinimidyl-2-(biotinamido)ethly-1,3-dithiopropionate (sulfo-NHS-SSbiotin) or, on occasion, sulfosuccinimidyl-6-(biotinamido)hexanoate
(sulfo-NHS-LC-biotin; Perbio, Tattenhall, UK), and then washed a
further three times in BBS.
Preparation of a PM-enriched Membrane Fraction—Biotinylated
cells were lysed in hypotonic lysis buffer (20 mM Tris-HCl, pH 7.5, 5
mM EDTA, 1 ␮l/107 cells protease inhibitor mixture) and subjected to
30 strokes of a tight fitting Dounce homogenizer. A PM-enriched
fraction was obtained from the hypotonic lysate according to the
method of Johnstone and Crumpton (23), with minor modifications.
Briefly, the Dounce homogenate was clarified by centrifugation
(4,000 ⫻ g, 15 min) to generate a postnuclear supernatant (PNS). The
PNS was centrifuged again (20,000 ⫻ g, 30 min) to yield a PMenriched 20,000 ⫻ g pellet (p20). Recovery of PM in the p20 was
between 50 and 80% as judged by the presence of the PM protein
linker for activation of T cells (LAT) or CD45 (data not shown). Aliquots
of the supernatant of the 20,000 ⫻ g spin (s20) and other cell fractions
were retained for protein analysis (see below). The p20 fraction was
solubilized (30 min) in hypotonic lysis buffer supplemented with 150
mM NaCl and 1% v/v Triton X-100. The choice of Triton X-100 on
economic grounds did not preclude the detection by Western blot of
the lipid raft markers, Thy1 (Fig. 2C) and LAT (data not shown) in the
solubilized p20 fraction. Insoluble material was pelleted (13,000 ⫻ g,
4 min), and biotinylated proteins were recovered from the supernatant
by incubation (overnight) with SA-agarose beads (2–10 ␮l 50% bead
slurry/107 cell equivalents). Beads were washed five times in ice-cold
RIPA buffer (10 mM Tris-HCl pH 7.5, 5 mM EDTA, 150 mM NaCl, 1%
w/v deoxycholate, 1% w/v Triton X-100, 0.1% w/v SDS), rinsed four
times in 1% Triton X-100, and eluted (1 h, room temperature) into
extraction buffer (8 M urea, 1% w/v Triton X-100, 1% w/v dithiothreitol) for IEF-based separations, or by boiling into 2⫻ SDS-PAGE
Molecular & Cellular Proteomics 3.1
57
Protein-MS Analysis of Plasma Membrane Proteins
sample buffer (125 mM Tris-HCl, pH 6.8, 0.025% w/v bromphenol
blue, 4% w/v SDS, 20% v/v glycerol, 5% v/v ␤-mercaptoethanol). All
procedures were performed at 4 °C unless otherwise stated. Cell
fractions were assayed for protein content prior to one-dimensional
PAGE and Western blotting using a bicinchonic acid assay kit
(Perbio).
Solution-phase IEF—Aliquots (50 ␮l of 1–2 ml) of affinity-purified
PM preparations were retained for analysis by one-dimensional gel
electrophoresis (1DE). The remainder was subjected to solutionphase IEF in a Rotofor cell (Bio-Rad, Hemel Hempstead, UK) according to the manufacturer’s instructions using a gradient of pH 3–10.
The buffer used was extraction buffer supplemented with 1% pH
3–10 ampholytes (Bio-Rad). Twenty fractions (⬇2 ml each) were
collected under vacuum and concentrated through 10-kDa molecular
mass filter tubes (Vivascience, Hanover, Germany). Samples were
further concentrated using an SDS-PAGE “clean-up” kit (Amersham
Biosciences, Little Chalfont, UK; catalogue no. 80-6484-70) comprising an acid precipitation step and organic solvent wash in conjunction
with proprietary reagents. Solubilized proteins were boiled in SDSPAGE sample buffer containing 5% ␤-mercaptoethanol and separated over laboratory-cast gradient gels (4 –12% acrylamide) and
silver stained as described by Shevchenko et al. (24). Comparative
one-dimensional analyses of precipitated and unprecipitated samples
demonstrated greater than 90% protein recovery (data not shown).
Two-dimensional PAGE—Protein samples were dissolved in a twodimensional sample buffer (7 M urea, 2 M thiourea, 1% w/v ampholytes, pH 3–10 (all Amersham Biosciences); 1% w/v ASB-14 (CalBiochem, Nottingham, UK); 1% w/v DTT, 10 mM Tris-HCl, pH 7.5)
utilized by Molloy et al. (25) for analysis of bacterial outer membranes.
The chaotropes (urea and thiourea) and novel aminosulphobetaine
zwitterionic detergent (ASB-14) in this buffer have been reported to
enhance solubilisation of membrane proteins (6, 7). Solubilized proteins were loaded into 18-cm linear pH 3–10 immobilized pH gradient
(IPG) strips by in-gel rehydration (overnight, room temperature). Strips
were subjected to IEF (70,000 Volt hours) in a Multiphor II unit and
resolved in the second dimension with 10% Tris/glycine polyacrylamide gels using a Hoeffer DALT horizontal gel aparatus (Amersham
Biosciences), originally described by Anderson and Anderson (46).
Mass Spectrometry—Tandem electrospray mass spectra were recorded using a quandrupole time-of-flight hybrid quadrupole/orthogonal acceleration time-of-flight spectrometer (Micromass, Manchester,
UK) interfaced to a Micromass CapLC capillary chromatograph. Samples were dissolved in 0.1% aqueous formic acid, and 6 ␮l was
injected onto a Pepmap C18 column (300 ␮m ⫻ 0.5 cm; LC Packings,
Amsterdam, NL), and washed for 3 min with 0.1% aqueous formic
acid (with the stream select valve diverting the column effluent to
waste). The flow rate was then reduced to 1 ␮l/min, the stream select
valve was switched to the data acquisition position, and the peptides
were eluted into the mass spectrometer with an acetonitrile/0.1%
formic acid gradient (5–70% acetonitrile over 20 min).
The capillary voltage was set to 3,500 V, and data-dependant
MS/MS acquisitions were performed on precursors with charge
states of 2, 3, or 4 over a survey mass range 540 –1200. Known
trypsin autolysis products and keratin-derived precursor ions were
automatically excluded. The collision voltage was varied between 18
and 45 V depending on the charge and mass of the precursor.
Product ion spectra were charge-state de-encrypted and de-isotoped
with a maximum entropy algorithm (Max Ent; Micromass). Proteins
were identified by correlation of uninterpreted tandem mass spectra
to entries in SwissProt/TREMBL using ProteinLynx Global Server
(versions 1 and 1.1; Micromass). One missed cleavage per peptide
was allowed, and the fragment ion tolerance was set to 100 ppm.
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Molecular & Cellular Proteomics 3.1
Carbamidomethylation of cysteine was assumed, but other potential
modifications were not considered in the first pass search. All matching spectra were reviewed manually, but in cases were the score
reported by ProteinLynx global server was less than 100, additional
searches were performed against the NCBI nonredundant database
using MASCOT, which utilizes a robust probalistic scoring algorithm
(47). Identifications based on a single matching peptide were verified
manually using the MassLynx program Pepseq (Micromass).
Western blotting—Matched amounts of protein from cell fractions
of resting or activated cells (5–20 ␮g/lane) were separated over
4 –12% Bis/Tris pre-cast gels (Invitrogen, Paisley, UK) and transferred
to nitrocellulose membranes. Membranes were blocked (overnight,
4 °C) in Tween/Tris-buffered saline (TTBS; 0.05% v/v Tween-20, 10
mM Tris-HCl, pH 7.5, 150 mM NaCl) supplemented with 4% bovine
serum albumin (TTBS-BSA). Membranes were probed with appropriate primary antibodies (1 h, room temperature) diluted in TTBS-BSA.
Membranes were washed (4 ⫻ 15min) in TTBS and incubated (1 h,
room temperature) with appropriate horseradish peroxidase (HRP)conjugated secondary antibodies in TTBS-BSA. Membranes were
washed (4 ⫻ 15 min in TTBS) and proteins detected by enhanced
chemiluminescence.
Two-color Fluorescence-activated Cell Sorting (FACS)—Cells were
washed twice in FACS buffer (10 mM PBS, pH 7.2, 2% w/v BSA,
0.05% w/v sodium azide) and incubated in the same buffer supplemented with mouse immunoglobulin (Ig) G (100 ␮g/ml, 1h, 4 °C). Cells
were then incubated (1h, 4 °C, 1/1000 dilution) with phycoerythrinconjugated monoclonal antibodies to B220 or CD3 alone or in combination with biotinylated antibodies (1/100) to either major histocompatibility complex (MHC) class II-(I-aq), CD45, or CD69 (all from BD
Biosciences, Cowley, UK) or a control antibody, biotinylated mouse
IgG. Cells were then washed and resuspended in SA-conjugated
fluorescein isothiocyanate (1/1000) for 1 h at 4 °C, then fixed in 2%
formaldehyde in 10 mM PBS, pH 7.2. Cells were acquired using a
Becton Dickinson LSR benchtop flow cytometer and data analyzed
using CellQuest software (BD Biosciences).
Antibodies—A rabbit polyclonal antibody to murine osteoclast inhibitory lectin (OCIL) was a generous gift from Dr. Matthew Gillespie
(St. Vincents Institute of Medical Research, University of Melbourne,
Australia). A rabbit anti-nicastrin antibody was purchased from
Chemicon International (Harrow, UK). A polyclonal goat antibody to
the product of the murine glucocorticoid-induced tumor necrosis
factor receptor-related gene (GITR) was purchased from R&D Systems (Abingdon, UK). Mouse monoclonal and rabbit polycolonal antibodies to glucose-regulated protein of 78 kDa (GRP78) were obtained from Bioquote Ltd. (York, UK). A rat monoclonal antibody to
Thy1.2 was prepared in-house from a hybridoma generated in the
laboratory of Dr. H. Waldmann (26). A rabbit polyclonal anti-LAT (UBI,
Milton Keynes, UK) and a goat polyclonal antibody to CD45 (Santa
Cruz Biotechnology Inc., Wiltshire, UK) were also used on occasion.
HRP-conjugated secondary antibodies to mouse, rabbit, goat, and rat
immunoglobulins were obtained from DAKO (Ely, UK).
Confocal Microscopy—Hybridoma cells were biotinylated as described above or left unlabelled. Cells were washed twice (BBS) and
once in PBS and allowed to adhere to quadruplicate wells of a glass
adhesion slide (Bio-Rad). Adherent cells were fixed in 3% formaldehyde in PBS (15 min, room temperature), rinsed in PBS, and, where
indicated, permeabilized in 0.1% Triton X-100 in PBS (30 min, 4 °C).
Cells were rinsed in PBS and blocked (10 mg/ml BSA in PBS, 30 min,
4 °C), then incubated with SA-Alexa (1/1000 in PBS/BSA, 30 min,
4 °C) from Molecular Probes (Leiden, The Netherlands). Cells were
rinsed in PBS and visualized using an UltraView LCI confocal microscopy suite (Perkin Elmer, Beaconsfield, UK).
Protein-MS Analysis of Plasma Membrane Proteins
FIG. 1. Biotinylation of plasma membranes using sulfo-NHS-SSbiotin. T cell hybridomas were left unlabelled (a and b) or subjected to
cell surface biotinylation using sulfo-NHS-LC-biotin (0.1 mg/ml; c and
d) or a water-soluble analogue sulfo-NHS-SS-biotin (0.1 mg/ml; e and
f). Adherent cells were permeabilized or not, as indicated, using 0.1%
Triton X-100. Biotin labeling was tagged using SA-Alexa 488. Confocal and bright-field images were generated using a Perkin Elmer
UltraView LCI microscopy suite. The data shown are representative of
three similar experiments.
RESULTS AND DISCUSSION
Our strategy was to label with biotin solvent-exposed lysine
residues on the surface proteins of intact cells (10) prior to
affinity purification of tagged proteins using SA-agarose
beads. To optimize the biotinylation conditions, we compared
two reagents, sulfo-NHS-LC-biotin (LC-biotin), recently used
in several similar studies (11–13), and a more water-soluble
analogue, sulfo-NHS-SS-biotin (SS-biotin). A SA-conjugated
fluorescent dye, Alexa 488 was used to visualize the subcellular distribution of labeled proteins (Fig. 1). In cells not treated
with biotinylation reagent (Fig. 1, a and b), no binding of
SA-Alexa was detected. In nondetergent-treated cells (Fig. 1,
c and e), biotinylation was confined to the plasma membrane.
However, in cells permeabilized to enable internalization of
SA-Alexa, biotinylation of the whole intracellular compartment
was apparent in a significant proportion (⬇30%) treated with
LC-biotin (Fig. 1d). In addition, ⬃30% of the cells labeled with
LC-biotin (Fig. 1d) and 56% of the cells labeled with SS-biotin
(Fig. 1f) exhibited a more restricted intracellular staining pattern, confined to clusters of subcellular structures. However,
the pronounced “whole cell” intracellular staining observed
with LC-biotin was not detected in any of the cells labeled
with SS-biotin. Accordingly, SS-biotin was used in subsequent studies.
FIG. 2. Enrichment of PM proteins by SA affinity purification. T
cell hybridomas were subjected to cell surface biotinylation and a
PNS prepared. Biotinylated proteins were harvested from a “PMenriched” membrane fraction (p20). Aliquots (8 ␮g total protein/lane)
of the PNS, p20, and SA affinity-purified material as well as the
post-affinity purification supernatant (post) and the supernatant of the
20 000 ⫻ g spin (s20) were subjected to 1DE. Proteins were transferred to nitrocellulose membranes that were probed as indicated.
The data are representative of three similar experiments.
Because some intracellular biotinylation was observed even
when the SS-biotin reagent was used it was not surprising
that SA affinity purification recovered a significant proportion
of abundant housekeeping proteins from whole-cell extracts
(data not shown). Such proteins (e.g. GRP78, actin) were not
detected by Western blotting if SA beads were incubated with
extracts of nonbiotinylated cells, confirming that their recovery was attributable to biotin/SA interactions (data not
shown). To minimize this contamination, we prepared a PMenriched fraction (p20) from biotinylated cells by differential
centrifugation. This fraction contained only 1–3% of the total
protein in a PNS but more than 50% of the PM markers CD45
and LAT (⬃25-fold enrichment, data not shown).
Biotinylated proteins were affinity-purified from this material
using SA-agarose beads, and equal amounts of protein (8 ␮g)
of the various fractions were probed for biotin with SA-HRP
(Fig. 2a) for the presence of PM proteins with anti-Thy-1 (Fig.
2c) and for non-PM proteins with an antibody to GRP-78, a
highly abundant endoplasmic reticulum lumen-located heat
shock protein (Fig. 2b). Compared with the PNS (lane 1) and
p20 (lane 2) fractions, the SA affinity-purified material (lane 3)
was enriched in biotinylated proteins and Thy-1 but depleted
of GRP78. After affinity purification, biotinylated proteins and
Thy-1 were largely absent from the p20 fraction, whereas
GRP78 content was unchanged (lane 4). Some proteins in the
Molecular & Cellular Proteomics 3.1
59
Protein-MS Analysis of Plasma Membrane Proteins
supernatant from the 20,000 ⫻ g spin (s20) were biotinylated,
though in many cases these appeared different to those enriched in the p20 and affinity-purified fractions (e.g. bands
arrowed in Fig. 2), suggesting that they may not be PM
proteins. Indeed, the PM marker Thy-1 was undetectable in
this fraction while GRP78 remained prominent. A similar distribution was observed using CD45 and actin as markers for
PM and non-PM proteins, respectively (data not shown).
These data demonstrate enrichment of PM proteins by
affinity purification, with concomitant depletion of intracellular
contaminants and suggest that almost all the p20 fractionassociated Thy-1 (Fig. 2) and CD45 (data not shown) are
biotinylated under these conditions and are completely recovered by affinity purification.
The observation that some intracellular proteins (Fig. 2a,
lane 5), including GRP78 (Fig. 2b, lane 3), were biotinylated
was consistent with the confocal microscopy results shown in
Fig. 1. However, we cannot exclude the possibility that the
small amount of biotinylated GRP78 detected was genuinely
exposed at the cell surface as has been reported (13).
We next used 2DE and MS/MS to catalogue the proteins
recovered in the affinity-purified fraction (27, 28). Equal
amounts of total protein from the PM-enriched p20 fraction
(5 ⫻ 107 cell equivalents) or the SA affinity-purified material
from the same cells (109 cell equivalents) were analyzed by
2DE. In each case, all detectable spots were excised and
subjected to MS/MS. In the former, 160 proteins were identified (molecular mass range 20 –150 kDa) of which seven
(4.4%) were PM proteins. Of the 17 proteins (molecular mass
range 25–180 kDa) identified in the SA affinity-purified material, five (29.4%) were PM proteins (see supplementary data
Fig. 1). In agreement with a similar recent study (13), several of
the non-PM species detected were heat shock proteins
(HSP70 and GRP78). One-dimensional SDS-PAGE of 20-fold
fewer cell equivalents (5 ⫻ 107) of the same SA affinitypurified sample led to the identification of 20 proteins (molecular mass range 24 –180 kDa), of which 11 (55%) were PM
proteins, some of which were not detected in either 2DE
experiment (see supplementary data Fig. 1). These latter included proteins that might be expected to perform poorly in
2DE, either because of their high molecular mass (e.g. CD45)
or basic pI (e.g. CD4, galectin 9). Thus, compared with 2DE,
one-dimensional SDS-PAGE appeared to favor identification
of PM proteins.
Most of the problems encountered in analyzing hydrophobic proteins by conventional 2DE are attributable to poor
solubility during IEF (which is incompatible with ionic detergents such as SDS), resulting in precipitation and inefficient
transfer from first to second dimension.
We therefore performed solution-phase IEF as the first dimension of separation using, instead of a polyacrylamide IPG
strip, a self-generating solution-phase gradient of pH 3–10.
Thus, affinity-purified PM proteins were focused into 20 discrete fractions, which were combined pairwise prior to SDS-
60
Molecular & Cellular Proteomics 3.1
FIG. 3. Resolution of PM proteins by solution-phase IEF. a, T cell
hybridomas were subjected to cell surface biotinylation, and biotinylated proteins were recovered (⬃1 ⫻ 109 cell equivalents) and separated by solution-phase IEF in a Rotofor cell (pH 3–10). Focused
proteins were recovered and subjected to one-dimensional PAGE.
Gels were silver stained and gel features excised and analyzed by
MS/MS. A representative tandem mass spectrum (b) of a peptide
derived from CD18 is shown.
PAGE on 4 –12% gradient gels (Fig. 3a), after which resolved
protein bands were excised and characterized by HPLC MS/
MS. The proteins so identified are listed in supplementary
data Table I (PM proteins) and supplementary data Table II
(“non-PM” proteins). A representative tandem mass spectrum, defining one of 18 peptide sequences matching integrin
␤2 (CD18), is shown (Fig. 3b). A total of 127 proteins were
identified from hybridoma T cells and unfractionated murine
splenocytes, of which 74 (58.3%) were PM proteins.
Many of the proteins identified were not detected by conventional 2DE and include high molecular mass (CD45, PTP␣),
hydrophobic (CD47, grand average of hydrophobicity
(GRAVY) score 0.563; tetraspanin 4-related protein, GRAVY
score 0.385), and basic species (galectin 9, CD4, EV12). This
contrasts with the 2DE analyses of the same samples in which
Protein-MS Analysis of Plasma Membrane Proteins
these proteins were not observed even though several (CD45,
CD4, galectin 9) were readily detected in one-dimensional
analyses of 20-fold fewer cell equivalents of the same material
(data not shown). Second, the use of solution-phase IEF
amplified the capacity of one-dimensional analyses from 5 ⫻
107 to ⬎2 ⫻ 109 cell equivalents of affinity-purified protein,
increasing the number of PM proteins identified in 11A2 cells
from 11 to 42.
The recovery of SIT, a type I membrane protein lacking
extracellular lysine residues, suggests that biotinylation of the
N-terminal amino group alone enables affinity capture. Thus,
theoretically, any N-terminally unblocked type I PM protein
could be isolated using this method.
While PM proteins constituted more than half of the total
identified, we also recovered a substantial number of proteins
normally associated with other cellular locations, (supplementary data Table II) as was predictable from earlier experiments
(Figs. 1 and 2). Given the punctate appearance of the intracellular staining depicted in Fig. 1, it is possibly noteworthy
that many of these “non PM” proteins have a role in protein
synthesis (translocon-associated protein ␦, oligosaccharyl
transferase, ribophorin, S60 ribosomal protein), folding
(GRP78, HSP70, HSP60, calreticulin, protein disulphide
isomerase), or trafficking (rab7, rab18, rab1B, SNAP23,
SNAP␣). This is consistent with the biotinylating reagent accessing the endoplasmic reticulum-Golgi complex and/or secretory vesicles, possibly via endocytosis notwithstanding the
low rate of endocytosis predicted at the temperature at which
labeling took place. Alternatively, the presence of some “nonPM” proteins may reflect in vivo interactions with the intracellular domains of PM proteins (e.g. Lck with CD4). There are
also reports that a small fraction of actin (29) and heat shock
proteins (13), including several identified here (e.g. HSP60,
HSP70, GRP78), may be expressed at the cell surface. Thus,
it is possible that their presence is not artifactual and simply
reflects their heterogeneous subcellular localization. However, other proteins (e.g. prohibitin, Bax, Bak, metaxin 2) are
mitochondrial. Their recovery probably reflects the contamination of the PM-enriched p20 fraction with mitochondrial
components (as documented in preliminary studies using prohibitin as a mitochondrial marker, data not shown) exposed to
the low levels of the biotinylation reagent gaining intracellular
access (see Fig. 1f).
Some of the proteins listed in supplementary data Table I
(e.g. CD98, basigin, lutheran antigen) have previously been
detected in proteomic studies of myeloid (19) or tumor cells
(13). Others, such as the “hypothetical” TM domain-containing protein of 11.4 kDa, had not previously been identified at
the protein level. Studies to examine its expression, localization, and function are now underway.
Several known proteins were detected in unfamiliar contexts in this study. Expression of nicastrin, for example, has
only recently been reported in human Jurkat T cells (30) and
its presence in primary splenocytes is, to our knowledge,
FIG. 4. Verification of solution-phase IEF data by immunoblotting. Murine splenocytes were incubated (16h, 37 °C) in the absence
(⫺) or presence (⫹) of PMA (30 ng/ml) and ionomycin (30 ng/ml). Cells
were subjected to cell surface biotinylation, and biotinylated proteins
were harvested and aliquots of other cell fractions retained. Aliquots
(5 ␮g total protein/lane) of each fraction were separated by onedimensional PAGE, transferred to nitrocellulose membranes, and
probed with the antibodies indicated. The data shown are representative of at least three similar experiments.
novel. Its expression was confirmed by Western blotting of
murine splenocytes (Fig. 4a) and 11A2 hybridomas (data not
shown). Nicastrin is a component of ␥-secretase (31, 32),
which generates the ␤-amyloid precursor protein implicated in
Alzheimer’s disease (33, 34). However, it is also involved in the
Notch signaling pathway (31, 35, 36), which may regulate T
and B cell development (37). Interestingly, the transmembrane
protease ADAM 10, also detected in our study (Table I), has
likewise been implicated in signaling by Notch (38).
Also noteworthy was the identification of a member of the
OCIL family of C-type lectins related to CD69 (39), originally
identified as an osteoblast-derived factor capable of potent
attenuation of osteoclast differentiation (40). Its expression on
murine splenocytes was confirmed by Western analysis (Fig
4b). OCIL may thus represent a potentially important component of the emerging regulatory circuit between the immune
system and the regulation of bone integrity, complementing
other regulatory molecules such as osteoprotegrin (41).
The rationale of these studies was to develop methods for
investigating differential expression of cell surface components. Thus, PM proteins were affinity-purified from primary
murine splenocytes with or without PMA/ionomycin treatment
(30 ng/ml, 16h), and equal amounts of protein were analyzed
by solution-phase IEF/SDS-PAGE. Levels of most proteins
were unchanged in resting and activated cells. However,
Molecular & Cellular Proteomics 3.1
61
Protein-MS Analysis of Plasma Membrane Proteins
quantitative comparisons were complicated by the presence
of a given protein in several Rotofor fractions and by the
presence of several proteins in some silver-stained bands.
TABLE I
Proteins regulated by cell activation
Unfractionated splenocytes were treated (16 h) or not with PMA/
ionomycin (30 ng/ml each), cell-surface biotinylated, and PM proteins
were recovered, resolved, and identified as described under “Experimental Procedures.” Proteins listed appeared up- or down-regulated
(as indicated) as judged by differential silver staining in resting and
activated cell extracts. Changes marked with an asterisk were selected for further study and subsequently confirmed by at least three
independent Western or FACS experiments.
Protein
Regulation by
PMA/ionomycin
IgM (membrane-bound form)
IgD (membrane-bound form)
Ig ␬ chain
MHC class 2 ␣ chain (I–Aq)
MHC class 2 ␤ chain (I–Aq)
GITR
Complement receptor-related protein
CD21 (complement receptor 2)
CD22
CD26
CD69
CD166 (ALCAM)
CD180
Down*
Down*
Down*
Up*
Up*
Up*
Up
Up
Up
Up
Up*
Up
Up
Notwithstanding these caveats, semiquantitative comparisons were possible between some bands that were clearly
differentially represented in the resting and activated samples
(Fig. 5 and Table I). The membrane-bound forms of IgM and
IgD (Fig. 5a), as well as Ig ␬ chain (Fig. 5e), presumably
derived from IgM and IgD, were down-regulated following
PMA/ionomycin challenge. In contrast, the Toll-like receptor,
CD180/RP105 (Fig. 5b), complement receptor-related protein
(Fig. 5c), GITR (Fig. 5d), and both the ␣ and ␤ chains of MHC
class II (Fig. 5e) were all up-regulated in the activated cells.
Because of the complicating factors alluded to above, a
selection of these changes were corroborated by Western
blotting (Fig. 4). Equal amounts of protein from extracts of the
same resting or activated cells were probed for four PM
proteins, nicastrin (Fig. 4a), OCIL (Fig. 4b), Ig ␬ chain (Fig. 4d),
and GITR (Fig. 4e). GRP78 (Fig. 4c) was used as a loading
control. As expected, all four PM proteins were enriched in the
p20 fraction compared with the PNS and further enriched in
the affinity-purified fraction. Levels of GRP78, OCIL, and
nicastrin in the affinity-purified PM preparation were unaffected by the PMA/ionomycin stimulus. In contrast, GITR was
clearly up-regulated and Ig ␬ chain was down-regulated by
activation, consistent with data in Fig. 5. The up-regulation of
MHC class II in activated cells was also confirmed (data not
shown).
FACS analysis was used independently to verify other
FIG. 5. PM protein expression following activation of murine splenocytes. Whole mouse splenocytes were incubated (16h, 37 °C) in the
absence (⫺) or presence (⫹) of PMA (30 ng/ml) and ionomycin (30 ng/ml). Resting and activated cells were cell-surface biotinylated, and labeled
proteins were recovered as described under “Experimental Procedures.” Recovered proteins from four independent experiments were pooled
and focused by solution-phase IEF in a gradient of pH 3–10. Fractions were processed and separated by one-dimensional PAGE as described
under “Experimental Procedures.” Proteins were visualized by silver staining and identified by MS/MS. See Table I. Two similar analyses of
smaller amounts of cell material yielded qualitatively similar results.
62
Molecular & Cellular Proteomics 3.1
Protein-MS Analysis of Plasma Membrane Proteins
FIG. 6. MHC class II (I-Aq) and CD69 are up-regulated in activated primary murine splenocytes. Unfractionated murine splenocytes were
incubated (16h, 37 °C) in the absence (filled panel) or presence (open panel) of PMA (30 ng/ml) and ionomycin (30 ng/ml). Resting and activated
cells were subjected to FACS analyses for CD69, MHC class II-I-Aq, or CD45 as described under “Experimental Procedures.” The data shown
are representative of three similar experiments.
changes observed in the affinity purification experiments. Activation-induced up-regulation of CD69 (Fig. 6a) and MHC
class II (I-Aq; Fig. 6b) were documented, whereas CD45 expression was unaffected (Fig. 6c). The Western and FACS
data together suggest that 3–5-fold expression differences were
detectable using the approach described here. Because OCIL,
GITR, and CD69 were each identified by MS/MS on the bases
of a single matching spectrum, expression of each molecule
was independently verified by Western or FACS experiments.
Several of these observations (see Table I), for example, the
up-regulation of MHC class II expression on murine B cells
and monocyte/macrophages, GITR on T cells, and CD69 on
both B and T cells, are known activation-induced changes.
Activation-associated increases in the expression of CD40
(42), CD166 (activated leukocyte cell adhesion molecule, ALCAM) (43), CD26 (dipeptidylpeptidase IV) (44), and CD180 (45)
have also been described. However, some of these changes
(e.g. complement receptor-related protein, a murine homologue of human complement receptor 1) have not to our
knowledge been reported previously. While the biological significance of these observations require further investigation,
the fact that six of 12 apparent activation-induced changes in
protein expression were corroborated by Western and FACS
analyses demonstrates that this method can be used to detect alterations in PM protein expression even when specific
reagents are unavailable.
Currently, this approach (in common with others that rely on
mass spectrometric detection) requires relatively large numbers of cells, which somewhat limits its application to in vivo
systems. However, because the method can detect markers
for given cell types within mixed populations (e.g. Thy-1 for T
lymphocytes, Ig receptor chains for B lymphocytes, CD11c
for monocytes), it could in principle be used to determine PM
protein expression profiles in mixed cell suspensions derived
from diseased tissues, such as tumors. In addition, the tech-
nique readily lends itself to nonsample-limited systems such
as analyses of PM protein expression on tumor cell lines.
In conclusion, by combining an optimized cell surface biotinylation procedure with solution-phase IEF, we have developed a generic and powerful strategy for profiling membrane
proteins that is easily implemented in most biochemical laboratories. In initial studies, we identified one unknown and 74
known transmembrane proteins, which is comparable to the
numbers detected in recent studies using multidimensional
liquid chromatography (14, 15, 19). We were also able to
detect differential protein expression in primary murine
splenocytes activated by treatment with PMA/ionomycin.
Thus, while we have not performed parallel shotgun analyses,
comparison of our results with published data suggest that
gel-based proteomic methods remain competitive with shotgun approaches. Both methods are capable of determining
differential expression profiles of membrane proteins in a
range of cell types under varying environmental and pathophysiological conditions.
Acknowledgments—We are grateful to Matthew Gillespie (St.
Vincents Institute of Medical Research, University of Melbourne,
Australia) for generous provision of antisera and to Katharina Speidel
and Parisa Amjadi for valuable help with FACS analyses.
* This work was supported by grants from the Arthritis Research
Campaign UK, The Medical Research Council UK, and the Wellcome
Trust. The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
□
S The on-line version of this article (available at http://www.
mcponline.org) contains supplemental data.
‡ To whom correspondence should be addressed: Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College
London, 1 Aspenlea Road, Hammersmith, London W6 8LH, United
Kingdom. Tel.: 44-20-8383-4444; Fax: 44-20-8383-4499; E-mail:
[email protected].
Molecular & Cellular Proteomics 3.1
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