Recent advances in ion mobility spectrometry (IMS) have illustrated its power in the structural characteristics of a molecule, especially when coupled with other separations dimensions such as liquid chromatography (LC) and mass spectrometry (MS). However, these three separation techniques together greatly complicate data analyses, so making better informatics tools are essential for assessing the resulting data. In this manuscript, Skyline was adapted to analyze LC-IMS-(CID)-MS data and determine the effect of adding the IMS dimension into the normal LC-MS molecular pipeline. For the evaluation, a tryptic digest of bovine serum albumin (BSA) was spiked into a yeast digest at 7 different concentrations, and calibration curves for both the precursor and all-ions fragments were analysesassessed with and without utilizing the IMS dimension. Skyline was able to rapidly analyze the MS and MS/MS data from 38 of the BSA peptides and in all cases the addition of the IMS dimension removed noise from interfering peptides resulting is in better calibration curves with higher correlation and lower limits of detection. This study presents an important informatics development since currently most LC-IMS-(CID)-MS data is studied manually and cannot be analyzed quickly. Since these evaluations require days for the analysis of only a few target molecules in a limited number of samples, it is unfeasible to evaluate hundreds of targets in numerous samples. Thus, this study showcases Skyline’s ability to work with multidimensional LC-IMS-(CID)-MS data and provide biological and environmental insights rapidly.

To evaluate the effect of utilizing the IMS dimension in multidimensional analyses and determine the performance of Skyline in analyzing the LC-IMS-(CID)-MS data, tryptically digested BSA was spiked at seven concentrations (100 pM, 1 nM, 5 nM, 10 nM, 100 nM, 500 nM, and 1 µM) into a tryptic yeast digest with a final peptide concentration of 0.1 µg/µL. Yeast was picked as the matrix of interest since most of the peptide components have a similar concentration range, providing more interfering peaks than samples with a higher dynamic range (i.e. plasma). MS1 and MS/MS all-ions fragmentation spectra were alternated every other second during each 100 minute LC run. Skyline was then utilized to extract the ion chromatograms and calculate peak areas for 38 different tryptic BSA target peptides in four ways including: LC-MS precursor extraction, LC-IMS-MS precursor extraction; LC-MS fragment extraction and LC-IMS-MS fragment extraction.

Bovine serum albumin (BSA) was purchased from Sigma-Aldrich (St. Louis, MO) and a tryptically digested yeast protein extract was purchased from Promega (Madison, WI). The BSA was tryptically digested and brought up to a concentration of 0.5 µg/µL in water. The BSA was then spiked into the tryptically digested yeast extract at 7 different concentrations (100 pM, 1 nM, 5 nM, 10 nM, 50 nM 100 nM, 500 nM, and 1 µM), where a final concentration of 0.1 µg/µL was utilized for the yeast peptides in all samples. A sample of BS peptides in water was also prepared at 100 nM to develop the Skyline parameters for the target peptides. Datasets for each sample were acquired by LC-IMS-MS using a Waters NanoAcquity HPLC system and an Agilent 6560 IM-QTOF MS platform (Agilent Technologies, Santa Clara) [1, 2]. The HPLC system utilized reverse phase columns prepared in-house by slurry packing 3 µm Jupiter C18 (Phenomenex, Torrence, CA) into 40 cm x x 360 µm o.d. x 75 µm i.d. fused silica (Polymicro Technologies Inc., Phoenix, AZ) using a 1-cm sol-gel frit for media retention. Trapping columns were prepared similarly by slurry packing 5-µm Jupiter C18 into a 4-cm length of 150 µm i.d. fused silica and fritted on both ends. Sample injections (5 µL) were trapped and washed on the trapping columns at 3 µL/min for 20 min prior to alignment with analytical columns. Mobile phases consisted of 0.1% formic acid in water (A) and 0.1% formic acid acetonitrile (B) and were operated at 300 nL/min with a 100-min gradient profile as follows (min:%B); 0:5, 2:8, 20:12, 75:35, 97:60, 100: 85. The LC was directly connected to the Agilent NanoESI source. Upon entering the source of the Agilent 6560 IM-QTOF MS platform, ions were passed through the inlet glass capillary, focused by a high pressure ion funnel, and accumulated in an ion funnel trap. Ions were then pulsed into the 78.24 cm long IMS drift tube filled with ~ 3.95 torr of nitrogen gas, where they travelled under the influence of a weak electric field (10-20 V/cm). Ions exiting the drift tube were refocused by a rear ion funnel and the collision energy was alternated between 0 V and 29 V for acquisition of MS and all ions fragmentation spectra. The ions were then detected with the TOF MS and their arrival time (tA) were recorded for the 100-3200 m/z range.