Catherine A. Srebalus Barnes

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 ∼900 to 1 200 and ∼3.7 to 4.6 × 105, 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.

Formation of peptide aggregates during ESI: Size, charge, composition, and contributions to noise

Ion mobility/time-of-flight techniques have been used to examine the onset of aggregation in model systems of Gly-Xxx (where Xxxx = Ala, Asn, Asp, Gln, Glu, His, Leu, Ser, Thr, and Trp) dipeptides. Under the experimental conditions employed, there is evidence that simple binary and quaternary mixtures of these dipeptides produce clusters containing as many as 16 to 75 peptide units (and 1 to 7 charges). In some systems, cluster compositions appear to come about largely from statistical association of peptide units; other dipeptide mixtures (and generally for small clusters) show evidence for nonstatistical behavior which could arise from some differences in gas-phase or solution thermochemistry. The minimum aggregate size appears to be largely determined by the charge state. Average thresholds for aggregate formation in the z = 2, 3, and 4 charge state families occur at m/z ∼500, 660, and 875, respectively. We briefly consider the idea that aggregates formed during electrospray ionization (ESI) may contribute to the background signal observed in the analysis of complex peptide mixtures.

Development of high-throughput liquid chromatography injected ion mobility quadrupole time-of-flight techniques for analysis of complex peptide mixtures.

The development of a multidimensional approach involving high-performance liquid chromatography (LC), ion mobility spectrometry (IMS) and tandem mass spectrometry is described for the analysis of complex peptide mixtures. In this approach, peptides are separated based on differences in their LC retention times and mobilities (as ions drift through He) prior to being introduced into a quadrupole/octopole/time-of-flight mass spectrometer. The initial LC separation and IMS dispersion of ions is used to label ions for subsequent fragmentation studies that are carried out for mixtures of ions. The approach is demonstrated by examining a mixture of peptides generated from tryptic digestion of 18 commercially available proteins. Current limitations of this initial study and potential advantages of the experimental approach are discussed.

Ion Mobility/Time-of-Flight Analysis of Combinatorial Library Mixtures

Recent developments in combinatorial synthesis and high-throughput screening (HTS) have emphasized parallel strategies for the generation and characterization of large libraries of synthetic molecules. In the era following the sequencing of the human genome [1], proteomics has been developed as a high-throughput approach to the discovery of biological target molecules that are up- or downregulated in diseased cells [2]. The identification of such targets will certainly increase the demand for combinatorial libraries of potential binding ligands that are chemically diverse, well characterized, and appropriate for screening against multiple target molecules.