Quantifying raft proteins in neonatal mouse brain by 'tube-gel' protein digestion label-free shotgun proteomics

1477-5956-5-17 1477-5956 Research Quantifying raft proteins in neonatal mouse brain by 'tube-gel' protein digestion label-free shotgun proteomics Yu Hongwei hyu@mcw.edu Wakim Bassam bwakim@mcw.edu Li Man manli@mcw.edu Halligan Brian halligan@mcw.edu Tint G Stephen tintgs@umdnj.edu Patel B Shailendra sbpatel@mcw.edu

Division of Endocrinology, Metabolism and Nutrition, Medical College of Wisconsin, Milwaukee, WI 53226, USA

Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA

National Center for Proteomics Research, Biotechnology and Bioinformatics Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA

Research Service, Department of Veterans Affairs New Jersey Health Care System, East Orange, NJ 07018, USA, and Department of Medicine, UMDNJ-New Jersey Medical School, Newark, NJ 07103-2714, USA

Department of Veterans Affairs, Clement J. Zablocki Medical Center, Milwaukee, WI 53295, USA

Qilu Hospital, Shandong University, 44 West Wenhua Road, Jinan, 250012, P. R. China

Proteome Science 1477-5956 2007 5 1 17 http://www.proteomesci.com/content/5/1/17 17892558 10.1186/1477-5956-5-17 24 5 2007 24 9 2007 24 9 2007 2007 Yu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

The low concentration and highly hydrophobic nature of proteins in lipid raft samples present significant challenges for the sensitive and accurate proteomic analyses of lipid raft proteins. Elimination of highly enriched lipids and interfering substances from raft samples is generally required before mass spectrometric analyses can be performed, but these procedures often lead to excessive protein loss and increased sample variability. For accurate analyses of the raft proteome, simplified protocols are needed to avoid excessive sample handling and purification steps.

Results

We have devised a simple protocol using a 'tube-gel' protein digestion that, when combined with mass spectrometry, can be used to obtain comprehensive and reproducible identification and quantitation of the lipid raft proteome prepared from neonatal mouse brain. Lipid rafts (detergent-resistant membranes using Triton X-100 extraction) prepared from neonatal mouse brain were directly incorporated into a polyacrylamide tube-gel matrix without prior protein separation. After in-gel digestion of proteins, nanospray LC-MS/MS was used to analyze the extracted peptides, and the resulting spectra were searched to identify the proteins present in the sample. Using the standard 'label-free' proteomics approach, the total number of MS/MS spectra for the identified proteins was used to provide a measure of relative protein abundances. This approach was successfully applied to lipid rafts prepared from neonatal mouse brain. A total of 216 proteins were identified: 127 proteins (58.8%) were predicted to be membrane proteins, or membrane-associated proteins and 175 proteins (~80%) showed less than a 2-fold variation in the relative abundance in replicate samples.

Conclusion

The tube-gel protein digestion protocol coupled with nanospray LC-MS/MS (TubeGeLC-MS/MS) offers a simple and reproducible method for identifying and quantifying the changes of relative abundances in lipid raft proteins from neonatal mouse brain and could become a useful approach for studying lipid raft proteins from various tissues.

Background

Lipid rafts are cholesterol- and sphingolipid-enriched specialized structures present in biological membranes 12345 that can be isolated by various techniques. A common method for the isolation of the rafts is to prepare detergent-resistant membranes (DRMs) by extraction with the nonionic detergent Triton X-100 at cold temperature. Recent interest in lipid rafts arises from observations that some membrane proteins appear to partition preferentially into raft domains, and may require this environment for their biological activity 45. Many previous studies have utilized two-dimensional gel electrophoresis (2DE) for proteomic profiling, but this method is limited by its lower sensitivity and it is often inefficient when analyzing raft proteins. Mass spectrometry (MS) has become a powerful tool for the analysis of complex protein mixtures. Proteomics profiling of either protein mixtures fractionated by 1DE or unfractionated protein mixtures by protease digestion and LC-MS/MS analysis has become increasingly popular. Peptides are identified by searching the resulting MS/MS spectra against protein sequence databases and protein presence is inferred from peptide presence. This general approach is referred to as 'top-down' or 'shotgun' proteomics. Several studies utilizing 1D gel filtration or in-solution protein digestion, combined with stable isotope labeling or label-free LC-MS/MS, have successfully profiled the protein composition and abundance in lipid rafts prepared from different biological sources 67891011121314. However, quantitation of changes in the raft protein abundance under various experimental circumstances remains a major challenge. A number of technical factors are critical for analytical reliability, such as sample quality, reproducibility of the raft preparations, quality of the chromatography system, and the performance of the mass spectrophotometer. The most pressing problems for lipid raft proteomic investigations are those involving sample preparation and handling. Lipid raft samples prepared by different methods are composed of highly enriched lipids and low concentrations of hydrophobic proteins. Raft preparations also contain many non-proteinaceous substances including exogenous reagents, such as salts, buffers and detergents employed for sample preparation. These highly enriched lipids and non-protein components, or contaminants can often interfere with proteome analysis and their removal is a critical step before any proteome analysis can be performed. Although the low protein concentrations in raft samples do not present a limitation for analysis, methods used for removing lipids and other interfering substances from raft samples can lead to excessive protein loss. Thus, the process of lipid raft preparation suitable for mass spectrometry is a major factor in the variability of data obtained by these powerful proteomic techniques.

For accurate analyses of the raft proteome, a robust protocol avoids excessive purification steps, each of which lead to additional protein losses, is desirable. To avoid protein loss during sample preparation for mass spectrometry, a 'tube-gel' protein digestion protocol was adopted in which the lipid raft samples were directly incorporated into a polyacrylamide tube-gel without electrophoresis 15. Detergents, lipids and other possible LC-MS/MS interfering materials in the raft samples are eliminated from the gel matrix while proteins are retained in the gel matrix. After the in-gel digestion of proteins, automated nanospray liquid chromatography tandem mass spectrometry (nanospray LC-MS/MS) is used to analyze the extracted peptides for protein identification. This protocol was used to analyze the protein profile of lipid rafts prepared from neonatal mouse brain. Neonatal mouse brain was chosen because there have been few proteomic studies of lipid rafts from neonatal brain 161718192021. Neonatal brain disorders are an important cause of mortality and morbidity contributing to the development of autism, cholesterol biosynthesis disorders, and a myriad of learning and developmental neurological and cognitive disabilities 222324252627. Developmental membrane defects have been postulated as one of the pathophysiological processes in these neonatal brain disorders. Additionally, the higher sterol content in brain tissue presents an additional challenge in preparing lipid raft samples for nanospray LC-MS/MS analysis.

Starting with limited amounts of frozen brain tissue, a total of 216 raft proteins were identified. Among the identified proteins, 127 (58.8%) were predicted to be plasma membrane (PM) or PM associated proteins including a number of authentic raft and/or GPI and lipid anchoring proteins, receptors, channel proteins, synaptic proteins, kinases, heterotrimeric G protein subunits, and some novel membrane proteins important for neurodevelopment. The major brain raft proteins, reported in previous investigations 8181928, were also identified as high abundance raft proteins in the present study. An advantage of this method is that it allows for raft proteins to be digested directly, dramatically reducing variations due to sample preparation prior to mass spectrometry. In this study, the standard 'label-free' proteomics approach in which total MS/MS spectral count is utilized to quantify the relative abundance of the identified proteins was used 29. The results showed that the variations of relative abundance in ~80% of the identified proteins in replicate samples were less than 2-fold, suggesting that the method is highly reproducible. This approach offers a simple and reproducible protocol for identifying and quantifying changes in the relative abundance of the lipid raft proteins from neonatal mouse brain and could become a useful method for studying lipid raft proteins from various tissues.

Results and Discussion

Characterization of lipid rafts by sucrose density gradient ultracentrifugation

Biochemical isolation of lipid raft membranes by gradient ultracentrifugation, as well as their subsequent analysis, is a useful and simple method to determine if membrane components are located in raft microdomains. The distributions of total protein, sucrose density, and contents of sterols, sphingomyelin (SM) and ceramide (Cer), as well as the lighting-scattering properties at 620 nm, for each of the sucrose gradient fractions are summarized in Figure 1. Buoyant low density fractions 2–4 (DRMs/rafts) had the greatest light-scattering properties at 620 nm, consistent with a high content of lipids, but the non-raft fractions 8–11 had little or no absorbance. Conversely, most of the recovered proteins were present in the non-raft fractions and the total protein in the lipid raft fractions was too low to allow for accurate measurement by conventional methods. The lipid raft fractions 2~4 were highly enriched in sterols (a mixture composed of ~60% cholesterol, ~40% desmosterol, and trace amounts of other sterol precursors such as 7-dehydrodesmosterol and lathosterol), SM, and Cer compared to plasma membranes. Further characterization of the known raft and the non-raft marker proteins in the sucrose gradients was performed by immunoblotting (Figure 2). Known raft proteins, such as caveolin-1 (cav-1), flotillin-1 (flot-1), contactin-1 (Cntn-1), annexin -VI (Anx-VI, Anx6A), GTP-binding protein αq (Gαq), and NAP-22, were present in the low-density fractions (fractions 2~4). Various accepted non-raft markers, such as β-COP (a Golgi marker), transferrin receptor (TfR) (a non-raft membrane marker protein), α-tubulin (a cytoskeletal protein), calnexin (an ER resident membrane protein), and ATP synthase (a mitochondrial protein), were only present in the high-density fractions. Collectively, these results reflect the typical biochemical profiles of lipid rafts from brain tissue 83031.

Figure 1

Biochemical characterization of sucrose density gradient fractions of neonatal mouse brain

Biochemical characterization of sucrose density gradient fractions of neonatal mouse brain. Panel A shows the distributions of protein and sucrose density in membrane fractions from sucrose gradients of neonatal brain. Panel B shows the light-scattering properties of each fraction by absorbance at 620 nm. The buoyant low density fractions 2~4 showed the greatest light-scattering properties at 620 nm, consistent with a high content of lipids. Panel C shows the distribution of sterols (cholesterol, desmosterol, lathosterol, and 7-dehydrodesmosterol (7DHD)) in each fraction from sucrose gradients of neonatal mouse brain and Panel D shows total sphingomyelin (SM) and ceramide (Cer).

Figure 2

Localization of known raft and non-raft marker proteins in sucrose gradients

Localization of known raft and non-raft marker proteins in sucrose gradients. Post-nuclear homogenates (PNH) from neonatal brain tissues were extracted using 1% of Triton X-100 (TX) and fractionated in 5–40% discontinuous sucrose-density gradient as described in Methods. Twelve fractions of each 1.0 ml were collected from the top to bottom. Twenty μg of PNH protein (H) and equal 30 μl of each fraction of gradient were subjected to immunoblotting with antibodies against indicated proteins.

Analysis of core raft proteome in neonatal mouse brain by TubeGeLC-MS/MS

The core protein composition of lipid rafts from neonatal mouse brain was determined by using a tube-gel protein digestion coupled with nanospray LC-MS/MS (TubeGeLC-MS/MS) analyses. The major benefit of this modification is that the raft proteins (usually in limiting quantities) are digested in a tube-gel matrix without fractionation and purification. Thus, sample losses are minimized compared to in-gel digestion based on SDS-PAGE. Moreover, inclusion of detergents (Triton X-100 and SDS. See Methods) can facilitate the effective solubilization and denaturation of hydrophobic lipid raft membrane proteins 3233. After proteins are incorporated into the tube-gel, the detergents, lipids and other interfering substances can be efficiently eliminated by extensive washing with acetonitrile prior to protein enzymatic digestion and subsequent nanospray LC-MS/MS analysis, without any significant loss of the proteins that are trapped in the gel matrix 15. This tube-gel approach has been successfully employed for high throughout mass spectrometric analysis of membrane proteins 15.

Two biological replicates (raft preparations from two neonatal brains), each with two MS technical replicates, were analyzed by TubeGeLC-MS/MS method. The peptides and corresponding proteins that were commonly identified in two biological samples (total 4 replicates) were considered as confident protein identifications. All identified proteins were then searched using UniProtKB/Swiss-Prot Release 52.3, TMHMM 2.0, and PubMed, to obtain information about their subcellular localization. The presence of predicted or verified transmembrane domains, glycosylphosphatidylinositol (GPI)-anchors and the lipid consensus sequences for myristoylation, pamitolyation, geranylgeranylation, farnesylation, and prenylation was used to classify proteins as either a membrane protein, or a membrane-associated protein 31. Identified proteins were also analyzed by UniProtKB for the predicted presence of these motifs in order to provide an additional criterion for the evaluation. The overall experimental results for lipid raft core proteome of neonatal mouse brain thus characterized are shown in Figure 3. The complete lists of identified proteins, categorized as plasma membrane (PM) or PM-associated proteins and non-PM proteins, are shown in Tables 1 and 2, respectively.

Table 1

Plasma membrane associated proteins identified in the lipid rafts of neonatal mouse brain

Acc. No.

Protein name

Pep ^a

% cov ^b

MW (KDa)

PTM ^c

Spectral count ^d

SD ^e

P12960

Contactin-1 (Neural cell surface protein F3)

35.03

113.4

GPI

174.0

4.0

Q91XV3

Neuronal axonal membrane protein NAP-22

66.52

22.1

Lipid anchor

125.0

0.6

P59216

G-protein G(o), alpha subunit 1

25.28

40.1

Lipid anchor

101.0

2.0

P14824

Annexin A6

41.43

75.8

99.0

22.0

Q8BLK3

Limbic system-associated membrane protein

17.94

38.0

GPI

66.0

1.0

Q80Z24

Neuronal growth regulator 1 (Neurotractin)

14.99

37.9

GPI

56.5

2.5

Q61330

Contactin-2

20.69

113.2

GPI

44.5

5.5

P38401

G-protein G(i), alpha-1 subunit

19.03

40.4

Lipid anchor

42.5

10.5

O08917

Flotillin-1

29.04

47.5

40.0

0.0

Q8BFZ9

SPFH domain-containing protein 2

11.30

38.9

38.0

1.2

Q9Z2S9

Flotillin-2 (Reggie-1) (REG-1)

22.22

41.7

36.5

1.5

P13595

Neural cell adhesion molecule 1, 180 kDa isoform

9.43

119.4

32.5

10.5

P38402

G-protein G(i), alpha-2 subunit

8.78

40.5

Lipid anchor

30.0

4.0

Q9Z1G4

Vacuolar proton pump subunit 1

9.43

96.5

28.5

0.5

Q62188

Dihydropyrimidinase-related protein 3 (DRP-3)

20.39

61.9

21.5

0.5

P27600

G-protein alpha-12 subunit (G alpha-12)

8.49

44.0

Lipid anchor

20.5

5.5

O70443

G-protein G(z), alpha subunit (G(x) alpha chain)

17.85

40.9

19.5

1.5

Q99PJ0

Neurotrimin

11.95

38.0

GPI

17.0

1.0

Q9WTR5

Cadherin-13

9.26

78.3

GPI

17.0

2.0

P21278

G-protein alpha-11 subunit

9.78

42.0

15.0

0.6

P51150

Ras-related protein Rab-7

19.90

23.5

Lipid anchor

14.5

0.5

Q8VDN2

Sodium/potassium-transporting ATPase alpha-1 chain

8.61

113.0

13.5

0.5

P32736

Opioid-binding protein/cell adhesion molecule

13.08

38.1

GPI

13.0

2.0

P21279

G-protein G(q), alpha subunit

13.64

41.5

Lipid anchor

13.0

0.0

P01831

Thy-1 membrane glycoprotein precursor (Thy-1)

26.71

18.1

GPI

12.5

0.5

P59729

Ras and Rab interactor 3

3.14

107.3

12.0

2.0

Q6TMK6

G-protein G(I)/G(S)/G(T) beta subunit

8.81

37.4

12.0

4.0

P06837

Neuromodulin (Axonal membrane protein GAP-43

18.14

23.6

Lipid anchor

11.5

0.5

Q6PIE5

Sodium/potassium-transporting ATPase alpha-2 chain

10.71

112.2

11.0

1.0

P62821

Ras-related protein Rab-1A

16.26

22.7

Lipid anchor

11.0

1.0

P53994

Ras-related protein Rab-2A

20.38

23.5

Lipid anchor

10.5

0.5

P38403

G-protein G(k), alpha subunit

13.64

40.6

Lipid anchor

10.0

2.0

Q8BKV1

Glypican-2 precursor

5.88

63.3

GPI

10.0

2.0

Q68FD5

Clathrin heavy chain

4.06

191.6

10.0

3.0

Q91X78

SPFH domain-containing protein 1

24.48

38.9

9.5

2.5

P39688

Proto-oncogene tyrosine-protein kinase Fyn

3.57

59.9

Lipid anchor

9.5

1.5

Q61735

Integrin-associated protein (IAP)

4.64

33.1

9.5

0.5

P97792

Coxsackievirus and adenovirus receptor homolog

6.04

39.9

Lipid anchor

9.5

1.5

Q9WUC3

Lymphocyte antigen Ly-6H precursor

12.32

14.67

GPI

9.5

1.5

Q6PIC6

Sodium/potassium-transporting ATPase alpha-3 chain

4.45

111.7

9.0

1.0

Q8BMT4

Leucine-rich repeat-containing protein 33

2.75

77.1

9.0

1.0

P48036

Annexin A5

21.07

35.8

9.0

1.0

P17182

Alpha-enolase

11.57

47.1

9.0

0.6

O08532

L-type calcium channel subunit delta

2.75

124.6

8.5

2.5

P63044

Vesicle-associated membrane protein 2 (VAMP-2)

35.09

12.7

8.0

0.0

P54227

Stathmin (Phosphoprotein p19)

14.97

17.3

8.0

1.0

P61027

Ras-related protein Rab-10

16.58

22.5

Lipid anchor

8.0

1.0

Q9QZF2

Glypican-1 precursor

8.99

61.4

GPI

8.0

2.0

Q3U1F9

Transmembrane phosphoprotein Cbp

7.48

46.5

Lipid anchor

7.0

0.6

Q69Z26

Contactin-4

2.15

117.5

GPI

7.0

1.0

Q9R0N7

Synaptotagmin-7 (Synaptotagmin VII) (SytVII)

7.71

45.5

6.5

0.5

Q9Z0P4

Paralemmin

10.73

41.6

Lipid anchor

6.5

1.5

O35454

Chloride channel protein 6 (ClC-6)

6.56

97.0

6.5

1.5

P51863

Vacuolar ATP synthase subunit d

6.86

40.3

6.0

0.6

P62814

Vacuolar ATP synthase subunit B, brain isoform

8.04

56.6

6.0

1.0

Q9ER00

Syntaxin-12

11.72

31.19

6.0

2.0

Q7SIG6

Development and differentiation-enhancing factor 2

2.84

106.8

6.0

1.0

Q6PHN9

Ras-related protein Rab-35

5.50

23.0

Lipid anchor

5.5

0.5

P63213

G-protein G(I)/G(S)/G(O) gamma-2 subunit

8.78

37.4

5.5

0.5

P14094

Sodium/potassium-transporting ATPase beta-1 chain

8.25

35.2

5.0

2.0

Q6PCX7

Repulsive guidance molecule A

6.62

50.0

GPI

5.0

2.0

P70296

Phosphatidylethanolamine-binding protein (PEBP)

28.65

20.7

5.0

3.0

P59108

Copine-2 (Copine II)

6.76

61.0

5.0

1.7

Q9WV55

VAMP-associated protein A

4.98

27.3

4.5

0.5

P46096

Synaptotagmin-1

5.71

47.4

Lipid anchor

4.5

1.5

Q9D1G1

Ras-related protein Rab-1B

16.50

22.2

Lipid anchor

4.5

0.5

P07356

Annexin A2 (Annexin II)

13.95

38.5

4.5

1.5

Q99KR6

RING finger protein 34

4.53

42.0

4.0

1.0

Q91V41

Ras-related protein Rab-14

12.62

23.9

Lipid anchor

4.0

1.2

P10852

4F2 cell-surface antigen heavy chain

5.14

58.3

4.0

0.6

P35279

Ras-related protein Rab-6A (Rab-6)

5.34

23.6

Lipid anchor

3.5

1.5

Q9CQD1

Ras-related protein Rab-5A

10.28

23.6

Lipid anchor

3.5

0.5

O35963

Ras-related protein Rab-33B

4.82

25.8

Lipid anchor

3.5

0.5

Q8K386

Ras-related protein Rab-15

5.21

24.3

Lipid anchor

3.5

1.5

P60764

Ras-related C3 botulinum toxin substrate 3

7.33

21.4

Lipid anchor

3.5

0.5

Q9QXL2

Kinesin family member 21A

1.14

186.53

3.5

0.5

Q8R4A8

G-protein G(s), alpha subunit

13.64

45.7

Lipid anchor

3.5

1.5

Q9JMB8

Contactin-6

3.21

113.8

GPI

3.5

2.5

Q65CL1

Alpha-3 catenin (Alpha T-catenin)

1.79

99.8

3.5

1.5

Q60547

Synaptonemal complex protein 3

6.01

27.1

3.0

0.6

Q9DAS9

G-protein G(I)/G(S)/G(O) gamma-12 subunit

22.86

78.7

Lipid anchor

3.0

1.0

P97449

Aminopeptidase N (Membrane protein p161)

1.87

109.7

3.0

1.0

Q9JHS3

Late endosomal/lysosomal Mp1-interacting protein

14.52

13.48

3.0

1.0

P31324

Prkar2b

4.11

46.04

3.0

1.5

P61264

Syntaxin-1B2 (Syntaxin 1B)

8.01

33.3

2.5

1.5

P35278

Ras-related protein Rab-5C

6.51

23.4

Lipid anchor

2.5

0.5

P63011

Ras-related protein Rab-3A

8.68

25.0

Lipid anchor

2.5

0.5

P97855

Ras-GTPase-activating protein binding protein 1

3.02

51.8

2.5

0.5

Q9CYH2

Protein C10orf58 homolog

5.99

24.4

2.5

0.5

P11505

Plasma membrane calcium-transporting ATPase 1

1.43

138.7

2.5

0.5

P05480

Neuronal proto-oncogene tyrosine-protein kinase Src

5.38

60.6

Lipid anchor

2.5

0.5

Q07310

Neurexin-3-alpha

4.11

174.0

2.5

0.5

O35136

Neural cell adhesion molecule 2

1.56

93.2

GPI

2.5

1.5

O89051

Integral membrane protein 2B

6.04

30.3

2.5

0.5

O08842

GDNF family receptor alpha-2

3.68

51.6

GPI

2.5

0.5

O08545

Ephrin-A3 precursor

8.60

21.2

GPI

2.5

0.5

Q9R1T7

Inducible T-cell co-stimulator (CD278 antigen)

8.54

225.30

2.5

0.5

P60879

Synaptosomal-associated protein 25

6.83

23.3

Lipid anchor

2.0

1.0

P80236

Ras-related C3 botulinum toxin substrate 1

18.42

8.8

2.0

1.0

P68404

Protein kinase C beta type

2.38

76.9

2.0

0.6

Q04690

Neurofibromin

3.81

319.6

2.0

0.6

Q60437

Insulin receptor substrate p53

6.15

57.64

2.0

0.6

Q61411

GTPase HRas

12.77

21.3

Lipid anchor

2.0

0.6

P51655

Glypican-4 precursor (K-glypican)

4.86

62.6

GPI

2.0

1.0

P23818

Glutamate receptor 1 (GluR-1)

2.21

101.57

2.0

0.6

Q8VBX4

C-type lectin domain family 4 member K

4.24

37.6

2.0

0.6

Q8JZW4

Copine-5 (Copine V)

2.53

65.6

2.0

1.0

Q8BLR2

Copine-4 (Copine IV)

2.70

62.4

2.0

0.6

Q02013

Aquaporin-1

7.49

28.66

2.0

1.2

Q07076

Annexin A7

3.46

49.9

2.0

1.7

P97429

Annexin A4 (Annexin IV)

10.09

35.9

2.0

0.6

Q9DBE8

Alpha-1,3-mannosyltransferase ALG2

3.86

47.4

2.0

1.0

P84078

ADP-ribosylation factor 1

6.15

20.6

2.0

1.0

Q6QIY3

Sensory neuron sodium channel

3.09

220.6

2.0

0.6

P49817

Caveolin-1

7.91

20.54

2.0

0.6

P18708

Vesicle-fusing ATPase

2.69

82.54

1.5

0.5

O70439

Syntaxin-7

5.41

29.8

1.5

0.5

P16546

Spectrin alpha chain, brain

1.24

274.7

1.5

0.5

Q9QZB0

Regulator of G-protein signaling 17

6.22

24.3

1.5

0.5

Q9JIR4

Regulating synaptic membrane exocytosis protein 1

6.07

179.7

1.5

0.5

Q05909

Receptor-type tyrosine-protein phosphatase gamma

9.72

161.2

1.5

0.5

P46638

Ras-related protein Rab-11B

9.72

24.5

Lipid anchor

1.5

0.5

P05696

Protein kinase C alpha type(PKC-alpha)

2.09

76.8