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Standardized sample preparation to reduce proteome complexity facilitates subsequent proteome analysis. Here we describe a robust sequential extraction method that enables simple fractionation of proteins in their native state according to their subcellular localization, yielding four subproteomes enriched in (a) cytosolic; (b) membrane and membrane organelle-localized; (c) soluble and DNA-associated nuclear and (d) cytoskeletal proteins. Efficiency and selectivity is demonstrated by morphological-, two-dimensional electrophoresis image-, immunological- as well as enzymatic-analysis. In pilot studies, subcellular redistribution of regulatory proteins was successfully measured.

Dedicated and specific sample preparation and adequate chromatographic resolution prior to MS are necessary for comprehensive and site-specific glycosylation analysis to compensate for high heterogeneity of protein glycosylation, low-abundance of specific glycoforms and ion-suppression effects caused by coelution of other peptides. This article describes a scheme for glycopeptide profiling, which comprises HILIC batch enrichment followed by complementary HILIC and RP-LC in 1-D and 2-D approaches. For reproducible and sensitive nano-LC/ESI-MS analysis, we used ZIC-HILIC and RP18e monolithic silica capillaries and assessed their retention characteristics and complementarity for glycopeptide separations. The experiments revealed that pre-enrichment of glycopeptides in combination with LC employing both phases considerably improves site-specific elucidation of glycosylation heterogeneity. Zwitterionic hydrophilic interaction liquid chromatography showed high capability to separate glycopeptides by their glycan composition, which coeluted on RP18e. By varying solvent conditions, retention can be well tuned, and efficient separations were achieved even in absence of any additives like salt or formic acid. RP18e facilitated glycopeptide separations with high peak capacity based on peptide sequence and degree of sialylation. Implementing both orthogonal and complementary phases in 1-D and 2-D LC setups was shown to significantly increase the number of different identified glycoforms and possesses great potential for comprehensive glycoproteomics approaches.

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A simple and flexible system, employing a column switching technique, has been designed to allow the analysis of peptides and proteins smaller than 15 kDa by molecular weight in filtered urine samples by performing a direct on-column injection utilising simultaneous sample clean-up and trace enrichment. The positively charged peptides and small proteins in the sample are attracted to the inner, negatively charged pore structure of the RAM-SCX column while the larger proteins and uncharged or negatively charged compounds are excluded. After preconditioning with the biological sample, large amounts of sample can be injected. Several important and adjustable parameters for the proper use of a RAM-SCX column are described and discussed. The main parameters being: i) the column is sensitive to sample overloading, which may result in drastic changes in the adsorption of peptides; ii) adsorption appears to be flow-rate and concentration dependent, as the sample molecules need time to penetrate into the internal pore structure in order to find complimentary orientated adsorption sites; iii) dilution and pH adjustment of sample during the loading process. The biocompatibility and proof-of-principle of this separation platform was demonstrated using human urine samples. Data are presented on repeatability as well as on the reproducibility of different synthesised batches of restricted access material (RAM).