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Shiseido's Protein Analysis and Micro-Column LC
Osamu Shirota, Ph.D.
Vice Chief Researcher
Laboratory of Chromatographic Technologies
Shiseido Research Center

Following the progress of genome analysis ranging from procaryote at the end of 20th century to various organisms, proteins have further attracted attention as a key to connecting genome information to vital functions. In order to demystify the processes leading to manifestation, information (two-dimensional electrophoresis, amino acid sequencing) obtained by many researchers is stored in a database, such as genome analysis results in the method of collectively analyzing proteins, proteome mix, thanks to the current information technology (networks, search function, etc.), and is used for daily research1.

Proteins and peptides forming proteomes are generally analyzed by gel electrophoresis or MALDI-TOF mass spectrometry, while liquid chromatography (LC) and a combination of ESI-mass spectrometry with LC play important roles in observing individual substances in detail. The micro-column (inner diameter less than 1 mm) is often used for this purpose.

Merits of the microcolumn
A certain amount of irreversible adsorption is inevitable when peptides or proteins of large molecular weights are injected into a column. A common experience when trace proteins are injected into a column of a size for general use is that nothing elutes from the column thereafter. In the microcolumn, using only trace amount of packing materials, the amount of adsorption is extremely small, facilitating analysis of trace amounts (pmol region or less) of samples. Another merit is that noise derived from the mobile phase is so small that target substances can be analyzed with high sensitivity, as the mobile phase flow rate flowing into an ion source is minute in LC-MS using the microcolumn.
Shiseido's microcolumn
Shiseido has prepared a microcolumn (inner diameter of 0.3 and 0.5 mm) in the newest product lineup, in the well-reputed CAPCELL PAK. The CAPCELL PAK microcolumns have substantially equal separation performances to that of the general-use columns and semi-microcolumns (Fig. 1). These microcolumns were developed mainly assuming application to peptide analysis under gradient conditions with acetonitrile.
What is essential to microcolumn analysis.
When using the microcolumn, for faithfully reflecting the separation attained in the column to the detector, or to the finally obtained analysis results (chromatogram), consideration must be given to the apparatus. As a tool for using the micro LC and NANOSPACE SI-2 series in the micro-column region, the 3040 micro LC organizer has been prepared.
【Fig. 1】  Microcolumn standard chart
【Fig. 1】 Microcolumn standard chart
The micro LC organizer comprises a microcolumn thermostat, a flow splitter, a manual injector stand, and an injection signal output button. As a unique characteristic, the thermostat part is designed to allow a high degree of freedom to allow freedom in tubing length or tubing angle for connecting to various detectors. As an example, by installing the micro LC organizer between the NANOSPACE and a mass spectrometer, and laying the necessary tubing, a micro LC system can be structured. An example of the apparatus and a peptide map of tryptic digests obtained from standard proteins are shown in the figure.
Microcolumn Organizer
Connected to Thermoelectron Mass Spectrometer
Shoiseido continues to run challenging samples with CAPCELL PAK microcolumns and NANOSPACE LC system with Microcolumn Organizer !
References
1 Proteomics Reviews 2001, edited by Micheal J. Dunn, Wiley-VCH Verlag GmbH, Weinheim, Germany, 2001
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