The use of gas-phase ion mobility separation in LC-MS based bottom-up proteomic experiments is described.
A data-independent acquisition approach was employed in combination with one and two dimensional reversed phase liquid
chromatography separations. The ion mobility component in the acquisition schema affords additional acquisition
specificity and improved peptide resolution in terms of system peak capacity. The additional acquisition specificity was
mainly achieved by means of the combined retention and drift time association of product to precursor ions and chemical
noise reduction. Experimental data from replicate experiments were utilized to determine the rate of increase for the number
of detected ions and deconvoluted accurate mass – retention time pairs and protein and peptide identifications as a
measure of peak capacity by varying mass spectral resolution, consideration of multidimensional separations and the incorporation
of ion mobility into the analytical schema. The presented data reveals a high degree of reproducibility at the
ion detection, peptide and protein level, when analyzed under identical conditions, with a concurrent average increase in
the number of identified peptides and proteins of 160% in the instance of mobility assisted acquisitions. Two-dimensional
separations independently improved the peptide and protein identification rates by another 260% and 320%, respectively.
Collectively, both separation techniques, reversed phase chromatography and ion mobility, increased the peak capacity
and number of peptides and proteins significantly. The applicability of the technique and instrument platform for qualitative
and quantitative proteomics studies will be demonstrated.
Keywords: Ion mobility, traveling wave ion guide, liquid chromatography, mass spectrometry, proteomics, data independent
analysis, two-dimensional, peak capacity
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Published on: 10 February, 2013
Page: [199 - 211]