Exploiting ion-mobility mass spectrometry for unraveling proteome complexity.

Ion mobility spectrometry-mass spectrometry (IMS-MS) is experiencing rapid growth in proteomic studies, driven by its enhancements in dynamic range and throughput, increasing the quantitation precision, and the depth of proteome coverage. The core principle of ion mobility spectrometry is to separate ions in an inert gas under the influence of an electric field based on differences in drift time. This minireview provides an introduction to IMS operation modes and a description of advantages and limitations is presented. Moreover, the principles of trapped IMS-MS (TIMS-MS), including parallel accumulation-serial fragmentation are discussed. Finally, emerging applications linked to TIMS focusing on sample throughput (in clinical proteomics) and sensitivity (single-cell proteomics) are reviewed, and the possibilities of intact protein analysis are discussed.

Insights in column packing processes of narrow bore and capillary-scale columns: Methodologies, driving forces, and separation performance - A tutorial review.

Having fundamental insights in the properties of stationary phases and in the driving forces during the column packing process, is crucial to obtain highly efficient and robust packed-bed column technologies for use in separation science. Here we discuss the properties of the most commonly-used stationary-phases, i.e., silica particulate materials, including fully-porous and core-shell silica particles and provide an overview of the most commonly used column hardware and available frit technologies. The different packing approaches that are considered are dry packing, high-pressure slurry packing, electrokinetic packing, and packing using centrifugal forces. In particular, sedimentation of particles in slurries and particle interaction during the packing process affecting the resulting sphere packing are discussed.

Development of Immobilized Enzyme Reactors for the characterization of the glycosylation heterogeneity of a protein.

The mapping of post-translational modifications (PTMs) of proteins can be addressed by bottom-up proteomics strategy using proteases to achieve the enzymatic digestion of the biomolecule. Glycosylation is one of the most challenging PTM to characterize due to its large structural heterogeneity. In this work, two Immobilized Enzyme Reactors (IMERs) based on trypsin and pepsin protease were used for the first time to fasten and improve the reliability of the specific mapping of the N-glycosylation heterogeneity of glycoproteins. The performance of the supports was evaluated with the digestion of human Chorionic Gonadotropin hormone (hCG), a glycoprotein characterized by four N- and four O-glycosylation sites, prior to the analysis of the digests by nanoliquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS). Firstly, the repeatability of the nanoLC-MS/MS was evaluated and a method to control the identification of the identified glycans was developed to validate them regarding the retention time of glycopeptides in reversed phase nanoLC separation. The repeatability of the digestion with trypsin-based IMER was evaluated on the same hCG batch and on three independent batches with common located glycans up to 75%. Then, the performance of the IMER digestions was compared to in-solution digestions to evaluate the qualitative mapping of the glycosylation. It has given rise to 42 out of 45 common glycans between both digestions modes. For the first time, the complementarity of trypsin and pepsin was illustrated for the glycosylation mapping as trypsin led to identifications on 2 out of 4 glycosylation site while pepsin was informative on the 4 glycosylation site. The potential of IMERs for the study of the glycosylation of a protein was illustrated with the comparison of two hCG-based drugs, Ovitrelle® and Pregnyl®.