Development of Orthogonal Separation Methods for 2D-HPLC (MS/MS) Analysis of Peptides.

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Martin Gilar, Petra Olivova, Amy A. Daly, and John C. Gebler[Waters]
Second Symposium on the Practical Applications of Mass Spectrometry in the Biotechnology and Pharmaceutical Industries, Practical MS
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Practical MS
2D-HPLC is expected to provide a greater peak capacity than a single-dimensional LC for proteome analysis However, the necessary condition for the success of 2D-HPLC separation is a good orthogonality of utilized chromatographic modes. The most popular 2D-HPLC approach for separation of peptides is a combination of strong cation exchange (SCX) and reversed-phase (RP) HPLC, although the alternative approaches have been described. In this work we evaluated a mutual orthogonality of SCX, size exclusion (SEC), reversed-phase, (RP), and hydrophilic interaction (HILIC) chromatography modes using a sample of ~250 tryptic peptides (five digested proteins). Retention times of the peptides in each LC mode were recorded and plotted into a normalized 2D graph, and cross-correlated. The orthogonality of separation was defined as a fraction of area covered by eluting peaks in 2D plots. The highest degree of orthogonality was observed for a combination of HILIC-RP, followed by SCX-RP. A good orthogonality was also achieved for the RP-RP combination, in which the pH of the running mobile phases was altered, rather than the stationary phase. While the SEC-RP combination provided a substantial orthogonality, a low peak capacity of the SEC mode made this setup less practical for 2D-HPLC. The consequences of separation selectivity, peak capacity, and mobile phase composition of investigated LC modes on 2D-HPLC-MS proteomic experiments are discussed.

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