Proteomic Analysis of Integral Plasma Membrane Proteins

Yingxin Zhao, Wei Zhang, Yoonjung Kho, Yingming Zhao

Research output: Contribution to journalArticle

213 Citations (Scopus)

Abstract

Efficient methods for profiling proteins integral to the plasma membrane are highly desirable for the identification of overexpressed proteins in disease cells. Such methods will aid in both understanding basic biological processes and discovering protein targets for the design of therapeutic monoclonal antibodies. Avoiding contamination by subcellular organelles and cytosolic proteins is crucial to the successful proteomic analysis of integral plasma membrane proteins. Here we report a biotin-directed affinity purification (BDAP) method for the preparation of integral plasma membrane proteins, which involves (1) biotinylation of cell surface membrane proteins in viable cells, (2) affinity enrichment using streptavidin beads, and (3) depletion of plasma membrane-associated cytosolic proteins by harsh washes with high-salt and high-pH buffers. The integral plasma membrane proteins are then extracted and subjected to SDS-PAGE separation and HPLC/MS/MS for protein identification. We used the BDAP method to prepare integral plasma membrane proteins from a human lung cancer cell line. Western blotting analysis showed that the preparation was almost completely devoid of actin, a major cytosolic protein. Nano-HPLC/MS/MS analysis of only 30 pg of protein extracted from the affinity-enriched integral plasma membrane preparation led to the identification of 898 unique proteins, of which 781 were annotated with regard to their plasma membrane localization. Among the annotated proteins, at least 526 (67.3%) were integral plasma membrane proteins. Notable among them were 62 prenylated proteins and 45 Ras family proteins. To our knowledge, this is the most comprehensive proteomic analysis of integral plasma membrane proteins in mammalian cells to date. Given the importance of integral membrane proteins for drug design, the described approach will expedite the characterization of plasma membrane subproteomes and the discovery of plasma membrane protein drug targets.

Original languageEnglish (US)
Pages (from-to)1817-1823
Number of pages7
JournalAnalytical Chemistry
Volume76
Issue number7
DOIs
StatePublished - Apr 1 2004
Externally publishedYes

Fingerprint

Cell membranes
Membrane Proteins
Proteins
Biotin
Purification
Proteomics
Cells
Streptavidin
Pharmaceutical Preparations
Actins
Buffers
Contamination
Salts
Monoclonal Antibodies

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Proteomic Analysis of Integral Plasma Membrane Proteins. / Zhao, Yingxin; Zhang, Wei; Kho, Yoonjung; Zhao, Yingming.

In: Analytical Chemistry, Vol. 76, No. 7, 01.04.2004, p. 1817-1823.

Research output: Contribution to journalArticle

Zhao, Yingxin ; Zhang, Wei ; Kho, Yoonjung ; Zhao, Yingming. / Proteomic Analysis of Integral Plasma Membrane Proteins. In: Analytical Chemistry. 2004 ; Vol. 76, No. 7. pp. 1817-1823.
@article{26d4310f07c042b8845294ee64b7c4cf,
title = "Proteomic Analysis of Integral Plasma Membrane Proteins",
abstract = "Efficient methods for profiling proteins integral to the plasma membrane are highly desirable for the identification of overexpressed proteins in disease cells. Such methods will aid in both understanding basic biological processes and discovering protein targets for the design of therapeutic monoclonal antibodies. Avoiding contamination by subcellular organelles and cytosolic proteins is crucial to the successful proteomic analysis of integral plasma membrane proteins. Here we report a biotin-directed affinity purification (BDAP) method for the preparation of integral plasma membrane proteins, which involves (1) biotinylation of cell surface membrane proteins in viable cells, (2) affinity enrichment using streptavidin beads, and (3) depletion of plasma membrane-associated cytosolic proteins by harsh washes with high-salt and high-pH buffers. The integral plasma membrane proteins are then extracted and subjected to SDS-PAGE separation and HPLC/MS/MS for protein identification. We used the BDAP method to prepare integral plasma membrane proteins from a human lung cancer cell line. Western blotting analysis showed that the preparation was almost completely devoid of actin, a major cytosolic protein. Nano-HPLC/MS/MS analysis of only 30 pg of protein extracted from the affinity-enriched integral plasma membrane preparation led to the identification of 898 unique proteins, of which 781 were annotated with regard to their plasma membrane localization. Among the annotated proteins, at least 526 (67.3{\%}) were integral plasma membrane proteins. Notable among them were 62 prenylated proteins and 45 Ras family proteins. To our knowledge, this is the most comprehensive proteomic analysis of integral plasma membrane proteins in mammalian cells to date. Given the importance of integral membrane proteins for drug design, the described approach will expedite the characterization of plasma membrane subproteomes and the discovery of plasma membrane protein drug targets.",
author = "Yingxin Zhao and Wei Zhang and Yoonjung Kho and Yingming Zhao",
year = "2004",
month = "4",
day = "1",
doi = "10.1021/ac0354037",
language = "English (US)",
volume = "76",
pages = "1817--1823",
journal = "Analytical Chemistry",
issn = "0003-2700",
publisher = "American Chemical Society",
number = "7",

}

TY - JOUR

T1 - Proteomic Analysis of Integral Plasma Membrane Proteins

AU - Zhao, Yingxin

AU - Zhang, Wei

AU - Kho, Yoonjung

AU - Zhao, Yingming

PY - 2004/4/1

Y1 - 2004/4/1

N2 - Efficient methods for profiling proteins integral to the plasma membrane are highly desirable for the identification of overexpressed proteins in disease cells. Such methods will aid in both understanding basic biological processes and discovering protein targets for the design of therapeutic monoclonal antibodies. Avoiding contamination by subcellular organelles and cytosolic proteins is crucial to the successful proteomic analysis of integral plasma membrane proteins. Here we report a biotin-directed affinity purification (BDAP) method for the preparation of integral plasma membrane proteins, which involves (1) biotinylation of cell surface membrane proteins in viable cells, (2) affinity enrichment using streptavidin beads, and (3) depletion of plasma membrane-associated cytosolic proteins by harsh washes with high-salt and high-pH buffers. The integral plasma membrane proteins are then extracted and subjected to SDS-PAGE separation and HPLC/MS/MS for protein identification. We used the BDAP method to prepare integral plasma membrane proteins from a human lung cancer cell line. Western blotting analysis showed that the preparation was almost completely devoid of actin, a major cytosolic protein. Nano-HPLC/MS/MS analysis of only 30 pg of protein extracted from the affinity-enriched integral plasma membrane preparation led to the identification of 898 unique proteins, of which 781 were annotated with regard to their plasma membrane localization. Among the annotated proteins, at least 526 (67.3%) were integral plasma membrane proteins. Notable among them were 62 prenylated proteins and 45 Ras family proteins. To our knowledge, this is the most comprehensive proteomic analysis of integral plasma membrane proteins in mammalian cells to date. Given the importance of integral membrane proteins for drug design, the described approach will expedite the characterization of plasma membrane subproteomes and the discovery of plasma membrane protein drug targets.

AB - Efficient methods for profiling proteins integral to the plasma membrane are highly desirable for the identification of overexpressed proteins in disease cells. Such methods will aid in both understanding basic biological processes and discovering protein targets for the design of therapeutic monoclonal antibodies. Avoiding contamination by subcellular organelles and cytosolic proteins is crucial to the successful proteomic analysis of integral plasma membrane proteins. Here we report a biotin-directed affinity purification (BDAP) method for the preparation of integral plasma membrane proteins, which involves (1) biotinylation of cell surface membrane proteins in viable cells, (2) affinity enrichment using streptavidin beads, and (3) depletion of plasma membrane-associated cytosolic proteins by harsh washes with high-salt and high-pH buffers. The integral plasma membrane proteins are then extracted and subjected to SDS-PAGE separation and HPLC/MS/MS for protein identification. We used the BDAP method to prepare integral plasma membrane proteins from a human lung cancer cell line. Western blotting analysis showed that the preparation was almost completely devoid of actin, a major cytosolic protein. Nano-HPLC/MS/MS analysis of only 30 pg of protein extracted from the affinity-enriched integral plasma membrane preparation led to the identification of 898 unique proteins, of which 781 were annotated with regard to their plasma membrane localization. Among the annotated proteins, at least 526 (67.3%) were integral plasma membrane proteins. Notable among them were 62 prenylated proteins and 45 Ras family proteins. To our knowledge, this is the most comprehensive proteomic analysis of integral plasma membrane proteins in mammalian cells to date. Given the importance of integral membrane proteins for drug design, the described approach will expedite the characterization of plasma membrane subproteomes and the discovery of plasma membrane protein drug targets.

UR - http://www.scopus.com/inward/record.url?scp=1842529218&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=1842529218&partnerID=8YFLogxK

U2 - 10.1021/ac0354037

DO - 10.1021/ac0354037

M3 - Article

C2 - 15053638

AN - SCOPUS:1842529218

VL - 76

SP - 1817

EP - 1823

JO - Analytical Chemistry

JF - Analytical Chemistry

SN - 0003-2700

IS - 7

ER -