Journal of Chromatography B, 782 (2002) 385–392 www.elsevier.com / locate / chromb Peak capacity of ion mobility mass spectrometry: Separation of peptides in helium buffer gas * Brandon T. Ruotolo, Kent J. Gillig, Earle G. Stone, David H. Russell Laboratory for Biological Mass Spectrometry, Department of Chemistry, Texas A&M University, College Station, TX 77843, USA Abstract Advances in the field of proteomics depend upon the development of high-throughput separation methods. Ion mobility-mass spectrometry is a fast separation method (separations on the millisecond time-scale), which has potential for peptide complex mixture analysis. Possible disadvantages of this technique center around the lack of orthogonality between separation based on ion mobility and separation based on mass. In order to examine the utility of ion mobility-mass spectrometry, the peak capacity ( f ) of the technique was estimated by subjecting a large dataset of peptides to linear regression analysis to determine an average trend for tryptic peptides. This trend-line, along with the deviation from a linear relationship observed for this dataset, was used to define the separation space for ion mobility-mass spectrometry. Using the maximum deviation found in the dataset (611%) the peak capacity of ion mobility-mass spectrometry is |2600 peptides. These results are discussed in light of other factors that may increase the peak capacity of ion mobility-mass spectrometry (i.e. multiple trends in the data resulting from multiple classes of compounds present in a sample) and current liquid chromatography approaches to complex peptide mixture analysis.  2002 Elsevier Science B.V. All rights reserved. Keywords: Peptides; Helium buffer gas 1. Introduction ence between two separation mechanisms [4], of the techniques employed will define the peak capacity The relevance of ion mobility mass spectrometry ( f ) of a technique. Peak capacity, or the number of (IM-MS) to the field of proteomics, where the signals that can reside in an area of two-dimensional central challenges include the high throughput analy- space [5], is the most common parameter used to sis of complex mixtures [1] and mapping protein– evaluate the applicability of a separation method to protein interactions in a proteome [2,3], depends on the analysis of complex mixtures [6]. For example, the analytical utility of IM-MS as compared to separation by reversed-phase high-performance liq- current two-dimensional separation methodologies. uid chromatography (RP-HPLC), which is based on For any multidimensional separation, such as liquid the partitioning of analytes between a hydrophobic chromatography–mass spectrometry (LC–MS) or stationary phase and a hydrophilic mobile phase, IM-MS, the orthogonality, or the degree of differ- exhibits good orthogonality to mass measurement (MS) [7]. The high-degree of orthogonality between LC and MS significantly enhances applications of the *Corresponding author. Tel.: 11-979-845-3345; fax: 11-979- technique to very complex peptide mixtures because 845-9485. E-mail address: russell@mail.chem.tamu.edu (D.H. Russell). LC–MS disperses signals over a large two-dimen- 1570-0232 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S1570-0232(02)00566-4