Isoelectric Focusing Nonporous RP HPLC: A Two-Dimensional Liquid-Phase Separation Method for Mapping of Cellular Proteins with Identification Using MALDI-TOF Mass Spectrometry

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• 作者：Daniel B. Wall ; Maureen T. Kachman ; Siyuan Gong ; Robert Hinderer ; Steven Parus ; David E. Misek ; Samir M. Hanash ; and David M. Lubman
• 刊名：Analytical Chemistry
• 出版年：2000
• 出版时间：March 15, 2000
• 年：2000
• 卷：72
• 期：6
• 页码：1099 - 1111
• 全文大小：507K
• 年卷期：v.72,no.6(March 15, 2000)
• ISSN：1520-6882

文摘

A novel two-dimensional liquid-phase separation methodwas developed that is capable of resolving large numbersof cellular proteins. The proteins are separated by pI usingisoelectric focusing in the first dimension and by hydrophobicity using nonporous reversed-phase HPLC in thesecond dimension (IEF-NP RP HPLC). Proteins weremapped using original software in order to create a proteinpattern analogous to that of the 2-D PAGE image. RPHPLC peaks are represented by bands of different intensity in the 2-D image, according to the intensity of thepeaks eluting from the HPLC. Each peak was collectedas the eluent of the HPLC separation in the liquid phase.The proteins collected were identified using proteolyticenzymes, MALDI-TOF MS and MSFit database searching.Using IEF-NP RP HPLC, ~700 bands were resolved in apI range from 3.2 to 9.5 and 38 different proteins withmolecular weights ranging from 12 000 to 75 000 wereidentified. In comparison to a 2-D gel separation of thesame human erythroleukemia cell line lysate, the IEF-NPRP HPLC produced improved resolution of low mass andbasic proteins. In addition, the proteins remained in theliquid phase throughout the separation, thus making theentire procedure highly amenable to automation and highthroughput. It is demonstrated that IEF-NP RP HPLCprovides a viable alternative to the 2-D gel separationmethod for the screening of protein profiles.