Capillary isoelectric focusing-based multidimensional concentration/separation platform for proteome analysis

被引:132
作者
Chen, JZ
Balgley, BM
DeVoe, DL
Lee, CS [1 ]
机构
[1] Univ Maryland, Dept Chem & Biochem, College Pk, MD 20742 USA
[2] Univ Maryland, Coll Life Sci, Mass Spectrometry Facil, College Pk, MD 20742 USA
[3] Univ Maryland, Dept Mech Engn, College Pk, MD 20742 USA
[4] Univ Maryland, Syst Res Inst, College Pk, MD 20742 USA
[5] Calibrant Biosyst, Rockville, MD 20855 USA
关键词
D O I
10.1021/ac034014+
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
An integrated proteome concentration/separation approach involving on-line combination of capillary isoelectric focusing (CIEF) with capillary reversed-phase liquid chromatography (CRPLC) is developed for providing significant analyte concentration and extremely high resolving power toward protein and peptide mixtures. Upon completion of analyte focusing, the self-sharpening effect greatly restricts analyte diffusion and contributes to analyte stacking in narrowly focused bands with a concentration factor of similar to240. In addition to analyte focusing, CIEF as the first separation dimension resolves proteins/ peptides on the basis of their differences in pI and offers greater resolving power than that achieved in strong cation exchange chromatography. The grouping of two highly resolving and completely orthogonal separation techniques of CIEF and CRPLC, together with analyte focusing and concentration, significantly enhances the dynamic range and sensitivity of conventional mass spectrometry toward the identification of low-abundance proteins. The CIEF-based multidimensional separation/concentration platform enables the identification of a greater number of yeast soluble proteins than methods presented in the literature, yet requires a protein loading of only 9.6 mug. This protein loading is 2-3 orders of magnitude lower than those employed by the reported non-gel-based proteome techniques. The distribution of a codon adaptation index value for identified yeast proteins approximates to that predicted for the entire yeast proteome and supports the capability of CIEF-based proteome separation technology for achieving comprehensive proteome analysis. By reducing the inner diameter of chromatography columns from 180 mum to 100 mum, the required protein loading is further decreased from 9.6 mug to 960 ng, illustrating the potential usage of this proteome technology for the analysis of protein profiles within small cell populations or limited tissue samples.
引用
收藏
页码:3145 / 3152
页数:8
相关论文
共 42 条
[1]   Cell sampling - Laser capture microdissection: Molecular analysis of tissue [J].
Bonner, RF ;
EmmertBuck, M ;
Cole, K ;
Pohida, T ;
Chuaqui, R ;
Goldstein, S ;
Liotta, LA .
SCIENCE, 1997, 278 (5342) :1481-&
[2]  
Chen JZ, 2002, ELECTROPHORESIS, V23, P3143, DOI 10.1002/1522-2683(200209)23:18<3143::AID-ELPS3143>3.0.CO
[3]  
2-7
[4]   Capillary isoelectric focusing: The problem of protein solubility [J].
Conti, M ;
Galassi, M ;
Bossi, A ;
Righetti, PG .
JOURNAL OF CHROMATOGRAPHY A, 1997, 757 (1-2) :237-245
[5]   SCREENING OF UMBILICAL-CORD BLOOD HEMOGLOBINS BY ISOELECTRIC-FOCUSING IN CAPILLARIES [J].
CONTI, M ;
GELFI, C ;
RIGHETTI, PG .
ELECTROPHORESIS, 1995, 16 (08) :1485-1491
[6]   Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology [J].
Gygi, SP ;
Corthals, GL ;
Zhang, Y ;
Rochon, Y ;
Aebersold, R .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2000, 97 (17) :9390-9395
[7]   Proteome analysis of low-abundance proteins using multidimensional chromatography and isotope-coded affinity tags [J].
Gygi, SP ;
Rist, B ;
Griffin, TJ ;
Eng, J ;
Aebersold, R .
JOURNAL OF PROTEOME RESEARCH, 2002, 1 (01) :47-54
[8]   Quantitative analysis of complex protein mixtures using isotope-coded affinity tags [J].
Gygi, SP ;
Rist, B ;
Gerber, SA ;
Turecek, F ;
Gelb, MH ;
Aebersold, R .
NATURE BIOTECHNOLOGY, 1999, 17 (10) :994-999
[9]   Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry [J].
Han, DK ;
Eng, J ;
Zhou, HL ;
Aebersold, R .
NATURE BIOTECHNOLOGY, 2001, 19 (10) :946-951
[10]   A personal view of molecular technology and how it has changed biology [J].
Hood, L .
JOURNAL OF PROTEOME RESEARCH, 2002, 1 (05) :399-409