<title>Identification of proteins from whole cell lysates: high-resolution time-of-flight mass spectrometry</title>

Abstract

Identification of whole cell proteins by 2D-PAGE/in-gel digestion combined with mass spectrometry has become routine, but the time required to prepare the gel, perform the separation, and process the gel for mass spectrometry analysis requires several days. To overcome the disadvantages of 2D-PAGE/in-gel digestion, we developed simple separation/in-solution digestion combined with high- resolution matrix assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry for analysis of whole cell proteins. High resolution MALDI TOF mass spectrometry can be used to analyze complex protein mixtures, thus complete protein separation is not required. Microtip column (C8) separation/in-solution digestion minimizes the time and effort required for sample preparation. Solvent-gradient elution from microtip column (C8) is used to fractionate the proteins and the number of samples that must be mass-analyzed is reduced tenfold. Therefore, significantly fewer numbers of mass spectra are used to identify major proteins in the whole cell lysates. The whole procedure from protein extraction to analysis of mass spectral data can be completed in a day and several hundreds proteins can be identified.