Multidimensional separations of complex peptide mixtures: a combined high-performance liquid chromatography/ion mobility/time-of-flight mass spectrometry approach

High-performance liquid chromatography (HPLC) has been combined with high-resolution ion mobility separations and time-of-flight mass spectrometry (MS) for the analysis of complex biomolecular mixtures such as those that arise upon tryptic digestion of protein mixtures. In this approach, components in a mixture are separated using reversed phase HPLC. As mixtures of peptides exit the column, they are electrosprayed into an ion mobility/time-of-flight mass spectrometer. Mixtures of ions are separated based on differences in mobilities through a buffer gas, and subsequently dispersed by differences in mass-to-charge (m/z) ratios in a mass spectrometer. The multidimensional approach is feasible because of the large differences in timescales of the HPLC (minutes), ion mobility (milliseconds), and time-of-flight (microseconds) techniques. Peak capacities for the two-dimensional liquid chromatography-ion mobility separations (LC-IMS) and three-dimensional LC-IMS-MS separations are estimated to be similar to 900 to 1200 and similar to3.7 to 4.6 X 10(5), respectively. The experimental apparatus and data acquisition considerations are described; data for a mixture of peptides obtained upon tryptic digestion of five proteins (albumin, bovine and pig; cytochrome c, horse; hemoglobin, dog and pig) are presented to illustrate the approach. (C) 2001 Elsevier Science B.V.