Potential of poly(styrene-<Emphasis Type="Italic">co</Emphasis>-divinylbenzene) monolithic columns for the LC-MS analysis of protein digests |
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Authors: | Michiel H M van de Meent Sebastiaan Eeltink Gerhardus J de Jong |
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Institution: | (1) Division of Biomedical Analysis, Department of Pharmaceutical Sciences, Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands;(2) Department of Chemical Engineering, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium |
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Abstract: | Two polystyrene-based capillary monolithic columns of different length (50 and 250 mm) were used to evaluate the effects of
column length on gradient separation of protein digests. A tryptic digest of a 9-protein mixture was used as a test sample.
Peak capacities were determined from selected extracted ion chromatograms, and tandem mass spectrometry data were used for
database matching using the MASCOT search engine. Peak capacities and protein identification scores were higher for the long
column with all gradients. Peak capacities appear to approach a plateau for longer gradient times; maximum peak capacity was
estimated to be 294 for the short column and 370 for the long column. Analyses with similar gradient slope produced a ratio
of the peak capacities of 3.36 for the long and the short column, which is slightly higher than the expected value of the
square root of the column length ratio. The use of a longer monolith improves peptide separation, as reflected by higher peak
capacity, and also increases protein identification, as observed from higher identification scores and a larger number of
identified peptides. Attention has also been paid to the peak production rate (PPR, peak capacity per unit time). For short
analysis times, the short column produces a higher PPR, while for analysis times longer than 40 min, the PPR of the 250-mm
column is higher. |
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