High Recovery HPLC of Lipid Raft Domains
with the Agilent mRP-C18 Column – A New
Proteomic Strategy for Enabling Membrane
Protein Identifications
Application Note

Proteomics

Authors
James Martosella, Nina Zolotarjova, Hongbin Liu,
Pat Perkins, and Susanne Moyer
Agilent Technologies, Inc.
2850 Centerville Road
Wilmington, DE 19808-1610
USA

Abstract
Membrane proteins play a critical role in cellular
processes and currently account for ~70% of all known
pharmaceutical drug targets. However, despite their biological importance and natural abundance, they remain
an under-represented subset of studied proteins withinproteomics. Lipid rafts are membrane microdomains thatare enriched in cholesterol and glycosphingolipids and it
is believed that proteins embedded in the membrane
localize either to the clusters of sphigolipids and cholesterol or to the intervening fluid regions of glycerolipids.
They are extremely important entities of the plasma membrane and have been implicated in diverse cellular
processes, including signal transduction, endocytic
events, such as viral entry, and cholesterol trafficking.
Yet, while analyses of raft function and composition continues to be of great interest, their study has limitations
due to the extremely hydrophobic nature of the raftdomains.
A robust proteomic workflow was developed for the identification of membrane proteins from human brain lipid
rafts using a novel macroporous reversed-phase column(mRP-C18) and optimized chromatographic conditions.
The column conditions and special chromatographic
material combine to provide high-protein recoveries,
excellent separations and reproducible chromatography.
The robust column methodology was used in a multidimensional separation strategy to identify membrane proteins found in lipid rafts. The lipid rafts were fractionatedby the mRP-C18 column and further resolved by gel electrophoresis. Unique bands were identified and analyzed
by mass spectrometry. Using this strategy we have identified 158 proteins (73 membrane) and 35 integral
membrane proteins) from 48 excised gel bands.

Introduction
Some of the most important cellular functions are
intrinsically tied to biological membranes. However, due to difficulties in the biochemical purification and structure/function analysis of this proteinclass, membrane proteins are still much less studied than soluble proteins. Lipid rafts, comprised ofenriched region of sphingolipids and cholesterol
are inherently more difficult to analyze. Yet, they
are extremely important subdomains of the plasma
membrane and are implicated in associating with avariety of proteins, especially those involved in cellsignaling. Traditionally, proteomic analyses ofcomplex protein samples involve the resolution ofproteins using two-dimensional gel electrophoresis(2DGE) followed by the identification of resolved
proteins by mass spectrometry (MS) or simply by
shotgun proteomics methods, which combines twodimensional liquid chromatography (LC) andMS. However, by either method, solubility andrecovery of proteins remains an obstacle. For
2DGE, many hydrophobic proteins are not solubilized in the nondetergent isoelectric focusing
sample buffer and solubilized proteins are prone toprecipitation at their isoelectric point. For shotgun
proteomic methods, as well as 2DGE, limited
dynamic range of detection is also at issue because
membrane proteins are typically lower in abundance when compared with soluble proteins. Asan alternative approach, reversed-phase highperformance chromatography has been used for
resolving membrane proteins and peptides and isused as a means to reduce sample complexity.
But, chromatography of high-molecular mass andhydrophobic proteins presents a myriad of challenges that often prohibit its use. Column chromatography can present obstacles for researchersand often 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.
In this study, a robust multidimensional separation
strategy for identifying membrane proteins associated with lipid rafts is presented. Using the Agilent
mRP-C18 column, we simultaneously separated
and delipidated lipid raft proteins and collected
fractions for downstream analysis by 1DGE and
MS. The mRP-C18 column with use of optimized
chromatographic conditions provided high protein
recovery and excellent reproducibility. Equally as
important the mRP-C18 and optimized chromatographic conditions enables on-column dissociationof the lipids from the membrane proteins and gives
researchers the opportunity to independently
evaluate the lipid fractions.

Experimental
The high-recovery mRP-C18 for separating proteins is a product from Agilent Technologies(Wilmington, DE). A 4.6-mm × 50-mm mRP-C18
column (part number 5188-5231) was used with anautomated Agilent 1100 LC system with an2autosampler equipped with a 900-µL injection
loop, quaternary pump and column heating at
80 °C. The reversed-phase separations of lipid
rafts were performed under a set of optimized conditions using a combined multisegmented 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 re-equilibrate the column.
Sample Preparation and Solubilization of Lipid Rafts andLoading onto the mRP-C18
The lipid raft-enriched fraction was prepared fromneurologically normal human brain STG (Superior
Temporal Gyrus) by detergent extraction on ice
and flotation on sucrose gradient. After homogenization, the sample was incubated in 1% Triton
TBS buffer for 30 minutes on ice. A discontinuous
sucrose gradient was formed in the centrifuge
tubes (Rotor JS - 24.38, Beckman) with the sample
in 40% sucrose on the top of 80% sucrose and overlayered with the 35% and 5% sucrose. The gradients were centrifuged for 42 hours at 103,900 × g.
During centrifugation, lipid rafts migrated to the
low-density region of the gradient due to their highlipid content. Low-density material floating at the
5%–30% interface was collected and centrifuged topellet the detergent-resistant fraction. Final pellets
were suspended in d.H2O, aliquoted, analyzed forprotein concentration by BCA protein assay andstored at –80 °C.
Prior to HPLC injection, the lipid raft fractions
required dissolution in formic acid to enable injection onto the column. As needed, 100-µL aliquotsof the lipid raft fraction were dried in a centrifugal
vacuum concentrator (Thermo-Savant, Millford,MA), resolubilized in 200 µL of 80% formic acidand briefly sonicated for 30 seconds in a waterbath. The samples were then redried 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 1 µg/µL
in 80% formic acid. HPLC injection amounts varied
from 200 µL–500 µL depending on the amount ofprotein needed for fraction processing or
SDS PAGE analysis.

Lipid Raft HPLC Fractionation

Electrophoretic Analysis

An Agilent 1100 HPLC equipped with a quaternary
pump, thermostatted autosampler, thermostatted
column compartment, and a thermostatted
analytical-scale fraction collector was used for
fractionating the samples. Due to the larger volumes required for complete injection of samples,the standard 100-µL needle and sample loop fromthe autosampler was replaced with a 900-µL
needle and metering device (Agilent part number
G1363A). HPLC fractions were automaticallycollected by time into 1.5-mL plastic tubes (partnumber 5188-5251) at 4 °C. The fractions werethen dried in a centrifugal vacuum concentrator(Thermo-Savant, Millford, MA) and stored at–80 °C for LC-MS/MS and SDS-PAGE 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 minute 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).

After lipid raft preparation and subsequent solubilization with formic acid, the lipid rafts were separated under high temperature RP conditions using
a combination of a multisegmented elution gradient of water (0.1% TFA)/ACN (0.08% TFA) and alinear-elution gradient of ACN (20% formic acid)/
2i-Propanol (Table 1). Fraction collection was performed by time, collecting 1-minute time slices
starting at 1.0 minute and continuing to70.0 minutes.
Table 1.

Lipid Raft Fractionation Multisegmented GradientFlow

0.75 mL/min.

Stoptime

70.0 min.

Posttime

30.0 min.

Column temp.

80.0 ºC

Solvent A

Water/0.1% TFA

Solvent B

ACN/0.08% TFA

Solvent C

ACN/20% Formic acid

Solvent D

2i-Propanol

Detection UV

280 nmPressure limit

250 barRecovery Conditions
To measure protein recovery, lipid rafts were fractionated on the Agilent mRP-C18 column under thegradient conditions shown in Table 1. The entire
column effluent from a 200-µg injection was collected. Blank runs were performed in the samemanner, however, with the column removed fromthe flow path. Blanks and column eluates werethen dried in a SpeedVac at medium drying temperatures overnight. The proteins were resolubilized in 6M Urea, 1% Triton X-100 and 0.25% acetic
acid. Samples were vortexed extensively to solubilize all protein and remove any material adheringto the tube walls. Protein concentrations were
determined using a BCA protein assay kit (Pierce,
Rockford, IL). Recovery data for the sample withand without the column installed were used toaccurately determine the absolute protein recoveries. Using the same recovery methods, similar
results have been obtained by fluorescence detection with use of the EZQ Protein Quantitation kit
from Invitrogen/Molecular Probes (part number
R33200). Recovery was also determined qualitatively by performing blank runs after each column
run. Chromatograms were monitored for the presence of ghost peaks after runs to establish thepresence of any protein and lipid carryover.
1D-LC-MS/MS

Gradient
Time
(min)
0
40
50
55
65
70
75
80

%B
20
50
100
100
0
0
0
20

%C
0
0
0
0
100
0
0
0

%D
0
0
0
0
0
100
100
0

Gel bands were digested with trypsin using
an Agilent in-gel digestion kit (part number
5188-2745). The digested peptides were extracted
and proteins were identified by LC-MS/MS analysis on an Agilent 1100 nano-LC and 1100 MSD trap
XCT. The digests were loaded onto a capillary
300SB-C18 RP column (Agilent, 0.075 mm id ×
150 mm, part number 5065-9911). Elution of peptide fragments was accomplished by RP gradient3

elution with buffer A; water/3.0% ACN(0.1% formic acid) and buffer B; water/90% ACN(0.1% FA). The XCT ion trap mass spectrometer
was operated in standard scan mode for MS analysis and in ultrascan mode for MS/MS. Forty-eight
gel bands were analyzed and MS data weresearched against SwissProt Human database(total 12015 entries), using Spectrum Mill
(Agilent) computer database search algorithm.
The gradient for the RP separations is as follows:
Time (min)
%B

0
5

5
12

40
35

50
65

60 62
100 100

Results and Discussion
The majority of currently used techniques to analyze complex membrane-protein samples employ
2DGE coupled with MS or tandem LC shotgun MS
methods. However, these techniques are limited
due to poor protein solubilities, low protein loads,
and losses associated with poor protein recoveries.

The Agilent mRP-C18 column and optimized chromatographic conditions provide desirable separation characteristics which demonstrate excellent
utility for use in proteomic workflows and offer
several advantages over standard RP columns andsystems for separating, and fractionating, complex
sample mixtures such as lipid rafts.
Figure 1 is representative of a RP separation ofhuman brain lipid rafts on a 4.6-mm × 50-mm
mRP-C18. The area of the chromatogram from
0–48 minutes represents the region of highest protein elution, while the elution profile past thisregion consists mainly of lipids. Gradient compositions, elution times, and elevated temperature,
combined with the novel macroporous column
material enabled on-column lipid dissociation andminimized irreversible binding effects from lipids,
cholesterol and proteins. The lipid elution profile
shown in Figure 1 was analyzed by ESI LC-MS/MS(data not shown) and contains significant amountsof phospholipids and sphingomyelins. On-column
delipidation allows researchers to minimize protein losses commonly associated with extraction
and precipitation methods.

Lipid elution
mAU

25

20

15

10

5

0
0

Figure 1.

4

10

20

30

40

50

60

Reversed-phase separation of 500-µg human brain lipid rafts from a 4.6-mm x 50-mm mRP-C18 column.
The shaded region past 48 minutes represents the area of highest lipid elution (Chromatographic run
elutes to 80 minutes, however, the region past 70 minutes is significantly off-scale due to the i-propanol
UV trace and extended lipid elution.)minUsing the combination of chromatography withelectrophoretic analysis can enable a more informative approach to define the efficiency of an
HPLC separation. We collected fractions from an
mRP-C18 lipid raft separation and evaluated thechromatographic efficiency on an SDS PAGE
(Figure 2). The electrophoretic analysis of the separation detailed excellent protein resolution from
0–48 minutes and showed later eluting lipids past
the 48.0 minute area which would not enter the gellanes (data not shown).

Fractions

1-10 11-15 16-20 21 22

23

24

25

26

27

28

29

36

41

42

43

44

45

46

47

30

31

32

33

34

35

kD
200.0
116.3
97.4
66.3
55.4
36.5
31.0
21.5
14.4
6.0

Fractions

37

38

39

40

kDa
200.0
116.3
97.4
66.3
55.4
36.5
31.0
21.5
14.4
6.0

Figure 2.

1D SDS PAGE of the lipid raft separation shown in Figure 1. Fractions were combined
based on earlier SDS PAGE. A total of 48 gel bands were excised for LC/MS analysis.

5High protein recovery is a critical parameter for
useful protein prefractionation strategies andenables reproducible chromatography. This is critical for the validation of biomarkers by allowing fora direct quantitation between control and diseased
samples and also is important for multiple sample
processing and run-to-run reproducibility. Recovery data for the lipid rafts samples is shown in
Table 2.
Table 2.

Recovery of Lipid Raft Proteins from the Agilent
mRP-C18 Column as Determined by BCA Assay andFluorescence Detection

Protein conc.
No column(µg)

Protein conc.
mRP-C18 recovery(µg)% recovery(N = 4)112130113.8 ± 10.9

Starting protein concentrations were measured
with column blanks (no column in the flow path).
Column runs were normalized against the blankruns under the same elution conditions. As shownin the table, the mRP-C18 column and optimized
HPLC operational conditions provided high recovery of injected sample. Two methods of analysis
gave complimentary high recovery data at approximately 114% to the normalized sample. Recovery

measurements after mRP-C18 consistently yieldedhigher protein concentrations than the blank runs
which had the column removed from the flow path.
We believe the increase in recovered protein was
the result of the on-column lipid dissociation thus
making the delipidated raft proteins more accessible for the assay.
Pre- and post-column hydrophobic peptide separations were performed to evaluate the column surface after multiple lipid raft separations. Standard
C18 reversed-phase stationary phases will irreversibly bind hydrophobic proteins and peptides
and cause shifts in retention behavior and losses
in total peak area. The changes are good indicatorsfor column efficiency and thus can help in determining protein recovery. An initial peptide separation was established on a new mRP-C18 column
and the retention times compared to a postpeptide separation after multiple lipid raft injections. As shown in Figure 3, the retention shifts
are nominal and peak areas remain unchanged. In
both pre- and post-column injections, peptide
peaks have retained their retention times within
accepted chromatographic error and show no
indications of change due to surface-boundproteins.

mAU
35

Initial peptide separation

30(new column)

10.23

25

17.73

12.43

23.58

9.39

20
15
10
5

8.32
23.25

0
_5
0

5

10

15

mAU
35

25

min

25

min

23.27

17.46

12.21

Post peptide separation

20

10.00

30
9.16

25
20
15
10

22.97

5

8.13

0
_5
0

Figure 3.

6

5

10

15

20

Pre-and post-column peptide separations for characterizing the mRP-C18 surface. Nominal retention
shifts and consistent peak shapes indicate that the mRP-C18 column surface has remained unchanged
after four consecutive 221-µg lipid raft injections.

Protein carryover displayed as chromatographic
peak ghosting is a qualitative indicator for poor
column recovery. With poor column recoveries,
blank injection column elutions will slowly bleed
proteins and require either subsequent blank runs
or column wash regimes to establish a steady baseline. As a final gauge to measure protein recovery,
consecutive lipid-raft injections were run followed
by a subsequent blank injection (blank run) to
qualitatively determine the presence of protein
carryover. As shown in Figure 4, pre- and postcolumn blank injections are the same and show no
indication of ghosting. Consecutive chromatographic runs prior to the blank injections are displayed in Figure 5. High protein recovery is
attributed to both the stabilized macroporous C18
column material and the optimized elution
conditions.

mAU

4

Prerun column injection
3
Postrun column injection
after 5_100 µg lipid raft separations
2

1

Baseline drift from ipa - formic acid0
0

Figure 4.

10

20

30

40

50

min

mRP-C18 new column blank and blank column injection after 5–100 µg lipid raft runs to qualitatively detailrecovery. The absence of peak ghosting indicates high protein recovery and enables continual column use formultiple sample processing.

7

Reproducibility would provide a measure of confidence to compare and validate potential protein
membrane biomarkers identified in lipid rafts.
Chromatographic reproducibility was examinedusing the preferred quaternary elution conditions
and elevated temperature. Separations of lipid
rafts from three injections were compared and areshown in Figure 5. The RP separations are reproducible and show no changes from run-to-run inretention times, apparent selectivity, or band
widths.
The mRP-C18 column was used to collect a totalof 70 reversed-phase fractions using the optimized
chromatographic conditions presented. After visualization (Figure 2) of the 1D SDS PAGE, 48 bands
were chosen and excised, in-gel digested and subsequently analyzed by 1D LC-MS/MS. The
mRP-C18 prefractionation strategy combined withthe subsequent resolution on a 1D PAGE enabled
the identification of 158 proteins associated withthe lipid raft. Of these identified, 73 proteins wereidentified as membrane proteins with 35 of thosebeing integral membrane proteins. Table 3 shows
the identifications for the integral membraneproteins as identified by GO Miner database.

Using the mRP-C18 column to prefractionate
human lipid rafts, we suggest a proteomic strategy
for identifying membrane proteins. This robust andhighly reproducible workflow has demonstrated
utility for increasing membrane protein identifications from a highly complex and hydrophobic lipid
raft sample and holds enormous potential for identifying membrane protein biomarkers in clinically
relevant sample types. In addition, on-column
delipidation offers researchers the ability to separate and analyze the lipid portion of the sample,
separate from the protein fractions and thus
allows for a more complete lipid analysis. Lipid
separation by the mRP-C18 column and the optimized elution conditions presented, eliminates
protein losses which may occur with more commonly associated techniques, such as solvent
extraction.
The mRP-C18 reversed-phase separation of lipid
rafts permitted total protein recoveries approaching quantitative, displayed-enhanced peak resolution, showed excellent reproducibility, and enabled
on-column lipid dissociation. The combination of
the Agilent mRP-C18 RP column and optimized
chromatographic conditions, with the subsequentgel separation, enables researchers to reduce
sample complexity and thus permit a higher
certainty for increasing protein identifications.

mAU

5

4

3

2

1

0
0

Figure 5.

8

10

20

30

40

50

Reproducibility of the mRP-C18 column as shown in five consecutive 100 µg lipid raft injections. Column
blanks were performed between each run, shown in Figure 4, and indicate no apparent protein carryover .

minTable 3.

Thirty-Five Human Brain Lipid Raft Associated Integral Membrane Protein Identifications from 48 1D SDS PAGE Bands
UniqueAccession protein
numberscore

Protein
MW

SpectraMean
intensity

Microsomal glutathione S-transferase 3 (EC 2.5.1.18)(Microsomal GST-3) (Microsomal GST-III)

O1488024.07

16516.4

258100000

Flotillin-1

O7595546.33

47355.5

419600000

Protein C8orf2 (UNQ2441/PRO9924/PRO5003)

O9490520.437839.8226800000

Thy-1 membrane glycoprotein precursor (Thy-1 antigen)(CDw90) (CD90 antigen)

P0421654.56

17934.8611150000000

Sodium/potassium-transporting ATPase beta-1 chain(Sodium/potassium-dependent ATPase beta-1 subunit)

P0502671.81

35061.510426000000

Dihydrolipoyl dehydrogenase, mitochondrial precursor (EC 1.8.1.4)(Dihydrolipoamide dehydrogenase) (Glycine cleavage system L protein)

P0962231.15

54150.5290100000

Protein name

Cytochrome c oxidase polypeptide VIc precursor (EC 1.9.3.1)

P0966926.54

8781.513303000000

ADP,ATP carrier protein, heart/skeletal muscle isoform T1(ADP/ATP translocase 1) (Adenine nucleotide translocator 1) (ANT 1)(Solute carrier family 25, member 4)P1223539.76

32933.5

316800000

Cytochrome c oxidase subunit IV isoform 1, mitochondrial precursor(EC 1.9.3.1) (COX IV-1) (Cytochrome c oxidase polypeptide IV)

P13073114.67

19576.8

6677100000

Neural cell adhesion molecule 1, 120 kDa isoform precursor(N-CAM 120) (NCAM-120) (CD56 antigen)

P1359224.62

83770.5253000000

Neural-cadherin precursor (N-cadherin) (Cadherin-2)

P1902221.63

99851.928740000

Vacuolar ATP synthase subunit B, brain isoform (EC 3.6.3.14)(V-ATPase B2 subunit) (Vacuolar proton pump B isoform 2)(Endomembrane proton pump 58 kDa subunit) (HO57)

P21281355.3556501

118166000000

Vacuolar ATP synthase subunit C (EC 3.6.3.14) (V-ATPase C subunit)(Vacuolar proton pump C subunit)

P21283161.3943941.823263000000

Voltage-dependent anion-selective channel protein 1 (VDAC-1)(hVDAC1) (Outer mitochondrial membrane protein porin 1)(Plasmalemmal porin) (Porin 31HL) (Porin 31HM)

P21796207.730641.5

10351100000

Oligodendrocyte-myelin glycoprotein precursor

P2351549.85

49608.2

4146000000

ATP synthase B chain, mitochondrial precursor (EC 3.6.3.14)

P2453981.95

28908.812139000000

ATP synthase alpha chain, mitochondrial precursor (EC 3.6.3.14)

P25705182.4659750.9

14131000000

Prohibitin

P3523241.63

29804.2

414200000

ATP synthase gamma chain, mitochondrial precursor (EC 3.6.3.14)

P3654217.39

32996.2237900000

Vacuolar ATP synthase subunit E (EC 3.6.3.14) (V-ATPase E subunit)(Vacuolar proton pump E subunit) (V-ATPase 31 kDa subunit) (P31)

P36543203.6326145.585

171000000

Vacuolar ATP synthase catalytic subunit A, ubiquitous isoform(EC 3.6.3.14) (V-ATPase A subunit 1) (Vacuolar proton pump alphasubunit 1) (V-ATPase 69 kDa subunit 1) (Isoform VA68)

P3860617.58

68304.5246900000

Voltage-dependent anion-selective channel protein 2 (VDAC-2)(hVDAC2) (Outer mitochondrial membrane protein porin 2)

P4588042.79

38092.9837300000

Dihydropyridine-sensitive L-type, calcium channel alpha-2/
delta subunits precursor

P5428947.68

123184434000000

Vacuolar ATP synthase subunit d (EC 3.6.3.14) (V-ATPase d subunit)(Vacuolar proton pump d subunit) (V-ATPase AC39 subunit)(V-ATPase 40 kDa accessory protein) (P39) (32 kDa accessory protein)

P6142146.33

40329.3

399100000

Contactin associated protein 1 precursor (Caspr) (Caspr1)(Neurexin 4) (Neurexin IV) (p190)

P78357318.22156267.542152000000

9

www.agilent.com/chem

Table 3.

Thirty-Five Human Brain Lipid Raft Associated Integral Membrane Protein Identifications from 48 1D SDS PAGE Bands(continued)
UniqueAccession protein
numberscore

Protein
MW

SpectraMean
intensity

Contactin 2 precursor (Axonin-1) (Axonal glycoprotein TAG-1)(Transient axonal glycoprotein 1) (TAX-1)

Q0224646.22113393.94118000000

Mitochondrial 2-oxoglutarate/malate carrier protein (OGCP)

Q0297825.01

33930.7228900000

Opioid binding protein/cell adhesion molecule precursor(OBCAM) (Opioid-binding cell adhesion molecule) (OPCML)

Q1498230.8

38007.8474600000

Myelin-oligodendrocyte glycoprotein precursor

Q1665354.63

28179.2

828500000Homer protein homolog 1

Q86YM7

84.34

40277

6

72500000

Vacuolar proton translocating ATPase 116 kDa subunit a isoform 1(V-ATPase 116-kDa isoform a1) (Clathrin-coated vesicle/synaptic
vesicle proton pump 116 kDa subunit) (Vacuolar proton pump subunit 1)(Vacuolar adenosine triphosphatase

Q9305046.42

96413.3528500000Toll-interacting protein

Q9H0E2

25.85

30282

2

18800000

Pleckstrin homology domain-containing protein family B member 1(Pleckstrin homology domain retinal protein 1) (PH domain
containing protein in retina 1) (PHRET1)

Q9UF11

27.3

27186.1

216600000

Bassoon protein (Zinc-finger protein 231)

Q9UPA526.43416370.1224100000

Vacuolar ATP synthase subunit D (EC 3.6.3.14) (V-ATPase D subunit)(Vacuolar proton pump D subunit) (V-ATPase 28 kDa accessory protein)

Q9Y5K8148.3128263

24105000000

Protein name

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