Nanowell-mediated multidimensional separations combining nanoLC with SLIM IM-MS for rapid, high-peak-capacity proteomic analyses

Abstract

Mass spectrometry (MS)-based analysis of complex biological samples is essential for biomedical research and clinical diagnostics. The separation prior to MS plays a key role in the overall analysis, with separations having larger peak capacities often leading to more identified species and improved confidence in those identifications. High-resolution ion mobility (IM) separations enabled by Structures for Lossless Ion Manipulation (SLIM) can provide extremely rapid, high-resolution separations and are well suited as a second dimension of separation following nanoscale liquid chromatography (nanoLC). However, existing sample handling approaches for offline coupling of separation modes require microliter-fraction volumes and are thus not well suited for analysis of trace biological samples. We have developed a novel nanowell-mediated fractionation system that enables nanoLC-separated samples to be efficiently preconcentrated and directly infused at nanoelectrospray flow rates for downstream analysis. When coupled with SLIM IM-MS, the platform enables rapid and high-peak-capacity multidimensional separations of small biological samples. In this study, peptides eluting from a 100 nL/min nanoLC separation were fractionated into ~ 60 nanowells on a microfluidic glass chip using an in-house-developed robotic system. The dried samples on the chip were individually reconstituted and ionized by nanoelectrospray for SLIM IM-MS analysis. Using model peptides for characterization of the nanowell platform, we found that at least 80% of the peptide components of the fractionated samples were recovered from the nanowells, providing up to ~tenfold preconcentration for SLIM IM-MS analysis. The combined LC-SLIM IM separation peak capacities exceeded 3600 with a measurement throughput that is similar to current one-dimensional (1D) LC-MS proteomic analyses. Graphical abstract A nanowell-mediated multidimensional separation platform that combines nanoLC with SLIM IM-MS enables rapid, high-peak-capacity proteomic analyses.

Keywords: Ion mobility; Mass spectrometry; Nanoelectrospray; nanoPOTS.

Fig. 1

Workflow for the nanowell-mediated nanoLC-SLIM IM-MS platform. (a) Samples are separated using LC, fractionated into nanowells and allowed to dry. (b) Samples in each nanowell are reconstituted and electrosprayed for subsequent IM-MS analysis. The insert images illustrate the reconstitution (1) and aspiration (2) process for reconstituting and collecting samples, and nanoESI (3) for subsequent SLIM IM-MS separation and analysis

Fig. 2

Sample recovery from nanowells using 10 μM Fluor 488-labeled [pSer5]-kemptide. Fluorescence images of original peptides (a) and remaining peptides (b) in nanowells after extraction, as well as their corresponding fluorescence intensities (c) (n = 3)

Fig. 3

Preconcentration effect of the nanowells. Effective concentration factors at different ratios of loading/reconstitution volume by using peptides leucine enkephalin and methionine enkephalin (n = 3)

Fig. 4

Nanowell platform for nanoESI-MS analysis. (a-b) Chromatography of reconstituted peptides leucine enkephalin (a) and methionine enkephalin (b) from nanowells for nanoESI-MS analysis (n = 6). (c) Recovery of the reconstituted peptides from nanowells

Fig. 5

Base peak chromatograph of triplicate nanoLC separations using 100 ng of HeLa digest