Optimizing hydrophilic interaction liquid chromatography for ultrasensitive proteome analysis

Abstract

In the last decade, the field of proteomics has rapidly progressed with substantial advances in many aspects, particularly nano-liquid chromatographic (LC) separation, mass spectrometric (MS) instrumentation and bioinformatics tools. However, significant improvements are still needed to generate proteome-wide data for limited number of cells. By enhancing the sensitivity of the LC-MS analysis, identification of lower abundant proteins can be achieved. The work described in this thesis highlights some relevant advances that can help accelerate proteomics towards a high level of depth in regard to proteome coverage and how these achievements can find ample application in several research lines. First, recent advances in peptide separation by multidimensional liquid chromatography are reviewed. The most common LC-based techniques employed in proteomics are reversed-phase (RP), ion-exchange (IEX) and hydrophilic interaction liquid chromatography (HILIC). A detailed overview of these separations, as well their combination in multidimensional formats, are provided. Then, we introduce and evaluate the use of HILIC-based strategies for the separation of complex peptide mixtures. Two zwitterionic stationary phases, ZIC-HILIC and ZIC-cHILIC, differing in the spatial orientation of their charged groups on the chromatographic material, are fully characterized with respect to separation efficiency and a number of peptide physico-chemical properties affecting peptide retention. We extensively tested the performances of these HILIC materials in 2D-LC strategy in combination with RP for the analysis of low amounts (few micrograms) of human cell digests showing that HILIC can rival traditional multidimensional strategies employed in proteomics. Next, we apply this 2D strategy to the analysis of a limited number of FACS-sorted colon stem cells. With an optimized sample preparation and workflow, we enabled for the first time the in-depth global proteome analysis of 10,000 colon stem cells, obtaining a high proteome coverage. Moreover, we describe the feasibility of the dimethyl labeling method in combination with ZIC-cHILIC technology for quantitative proteomics. We address the potential issue of isotope effects perturbing the essential co-elution of differently labeled peptides under ZIC-cHILIC separation. The deuterium effect can be largely eliminated by choosing appropriate pH conditions, and an optimized approach at pH 3 is a suitable quantitative strategy. Furthermore, we combine the HILIC separation with a phosphopeptide enrichment approach based on Ti4+-IMAC to develop an efficient approach with the aim of maximizing the coverage of the cellular phosphoproteome. We design and systematically compare three strategies including: a sole Ti4+-IMAC enrichment; Ti4+-IMAC enrichment followed by HILIC fractionation; a pre-fractionation based on strong cation exchange, followed by Ti4+-IMAC enrichment and a further step of fractionation by HILIC. This work demonstrates the need to carry out extensive fractionations for deep mining of the phosphoproteome and shows that the choice of appropriate analytical strategies mainly depends on sample amount and/or desirable analysis time. Finally, we summarize our expertise in the HILIC separation field into a protocol for sensitive shotgun proteomics. We report on our latest robust set-up for a ZIC-cHILIC chromatographic separation in a two-dimensional format. We give advice and propose practical solutions to issues that can be encountered during the development of a HILIC-based analytical strategy.