Coupling Ion Mobility Separations, Collisional Activation Techniques, and Multiple Stages of MS for Analysis of Complex Peptide Mixtures

Abstract

An ion trap/ion mobility/quadrupole/collision cell/time-of-flight mass spectrometer that incorporates a differentially pumped orifice-skimmer cone region at the back of the drift tube has been developed for the analysis of peptide mixtures. The combined approach allows a variety of strategies to be employed for collisionally activating ions, and fragments can be monitored by subsequent stages of mass spectrometry in a parallel fashion, as described previously (Anal. Chem. 2000, 72, 2737). Here, we describe the overall experimental approach in detail. Applications involving different aspects of the initial mobility separation and various collisional activation and parallel sequencing strategies are illustrated by examining several simple peptide mixtures and a mixture of tryptic peptides from beta-casein. Detection limits associated with various experimental configurations and the utility for analysis of complex systems are discussed.

Differentiation of Compact and Extended Conformations of Di-Ubiquitin Conjugates with Lysine-Specific Isopeptide Linkages by Ion Mobility-Mass Spectrometry

“…Nested ion mobility/timeof-flight mass spectrometry measurements were carried out using a home-built instrument at Indiana University. While a number of instrumental designs have been described [37,38] (and are likely to be valuable for these types of studies), the instrument used for these studies is a low-pressure drift tube that utilizes a drift region created by a series of insulating and conducting spacers which, when compressed, make the cavity that contains the buffer gas.…”

Modeling the resolution and sensitivity of FAIMS analyses

“…IMS has now been coupled to quadrupole MS [10], time-of-flight MS [13], and FTICR [14]. The IMS/TOF hybrid that disperses analyte mixtures in mobility and mass dimensions simultaneously is a promising new platform for high-throughput analytical applications [15,16].…”

Nanoflow LC/IMS-MS and LC/IMS-CID/MS of protein mixtures

“…Reviews of IMS methods are given elsewhere [25,[33][34][35][36] and the overall approach associated with IMS-TOF measurements has been described [37][38][39][40]. A schematic diagram of the instrument that was used in these studies is shown in Figure 1.…”

“…In the latter case, fragmentation occurs on a timescale that is much shorter than residence times of ions in the first field region of the drift tube. Therefore, it is possible to correlate fragments with precursor ions because they have the same total drift times, as described previously [39]. Upon exiting the drift tube, precursor ions (or precursors and fragments) are focused into the source region of a reflectron geometry TOF mass spectrometer and dispersed again according to differences in m/z ratios.…”

“…We refer to this as a "nested" technique because flight times are recorded on s timescales, within individual drift time windows across the ms timescale of the mobility separation; this has been described previously [37,39]. In addition, LC separations in this study occur on the order of seconds to minutes.…”