Octadecylated Silica Monolith Capillary Column with Integrated Nanoelectrospray Ionization Emitter for Highly Efficient Proteome Analysis

Abstract

An improved strategy for the preparation of octadecylated silica monolith capillary column with high homogeneity was proposed. Column performance was evaluated by nanoscale HPLC. The design for constructing an integrated nanoelectrospray emitter on the octadecylated silica monolith capillary column was first introduced. In comparison with the separated configuration where the emitter is connected to monolithic capillary column by the aid of a zero dead volume union, the integrated capillary column has the inherent advantage of the minimized extracolumn volume thus providing improved separation quality. The performance of the integrated monolithic capillary column was evaluated by separation of BSA tryptic digest, and peak capacity of 313 with a 30-cm column was obtained. The high separation performance allowed highly confident identification of 662 distinct proteins through assignment of 1933 unique peptides by analysis of tryptic digest of 0.5 g of Saccharomyces cerevisiae proteins. The higher separation efficiency by a 60-cm monolithic capillary column increased the proteome coverage with identification of 1323 proteins through assignment of 5501 unique peptides over 400-min gradient elution. Molecular & Cellular Proteomics 5:454 -461, 2006.The ultimate goal of proteomics is to study biological processes comprehensively by the systematic analysis of the proteins expressed in living systems (1). The basis for proteome analysis requires resolution of proteins followed by identification of the resolved proteins. Separation by twodimensional PAGE is the most widely used methodology in proteomic research (2, 3) as it combines two orthogonal separations to obtain efficient resolution of complex protein mixtures. However, two-dimensional PAGE has limitations such as difficulty to be automated and incompatibility with proteins of extreme pI values and molecular weight, low abundance proteins, and membrane-associated or -bound proteins (4 -6). Recently shotgun methodology based on nanoscale HPLC (nano-HPLC) 1 has emerged as an attractive alternative for proteome analysis because of its speed, ease of automation, and compatibility with mass spectrometry (7,8).Separation columns for nano-HPLC are usually fabricated by packing particulate beads with a controlled range of diameters and pore size. Particles of smaller diameters are preferably used to achieve a better efficiency, although they are hindered by the increase of backpressure. Recently the concept of monolithic column has been well established in separation technology (9 -13). The monolithic structure eliminates the interstitial voids in particulate columns, thus leading to fast mass transfer kinetics during separation. Moreover the enhanced permeability of monolithic rods results in a much lower backpressure, enabling the choice of longer columns to achieve increased separation performance (14,15). The pores present in the monolithic columns are key parameters affecting separation and permeability. As recommended by IUPAC, pores less than 2 nm in diameter are te...

Method Development of Efficient Protein Extraction in Bone Tissue for Proteome Analysis

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Automation of nanoflow liquid chromatography‐tandem mass spectrometry for proteome and peptide profiling analysis by using a monolithic analytical capillary column

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