Towards data acquisition throughput increase in Fourier transform mass spectrometry of proteins using double frequency measurements

Yury Tsybin, Aleksey Vorobyev
Elsevier, 2011
Article

Combined with high and ultra high pressure liquid chromatography, mass spectrometry-based peptide and protein structure analysis requires high resolution and mass accuracy achievable within short ion detection time. Although Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) offers highest resolution and mass accuracy among all types of mass spectrometers, it is unacceptably slow for many applications in proteomics requiring on-line sample pre-fractionation. Multiple frequency detection promises to increase the speed of ion detection in FT-ICR MS without the loss of resolving power, but its application to peptide and, especially, protein analysis has been negligible. In this report, we show that double frequency detection in high field FT-ICR MS indeed allows faster acquisition of high resolution mass spectra of proteins. (C) 2010 Elsevier B.V. All rights reserved.

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Yury Tsybin, Aleksey Vorobyev
Elsevier, 2011
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https://infoscience.epfl.ch/record/168784?ln=fr

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Extended Bottom-Up Proteomics with Secreted Aspartic Protease Sap9

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We investigate the benefits and experimental feasibility of approaches enabling the shift from short (1.7 kDa on average) peptides in bottom-up proteomics to about twice longer (similar to 3.2 kDa on average) peptides in the so-called extended bottom-up proteomics. Candida albicans secreted aspartic protease Sap9 has been selected for evaluation as an extended bottom-up proteomic-grade enzyme due to its suggested dibasic cleavage specificity and ease of production. We report the extensive characterization of Sap9 specificity and selectivity revealing that protein cleavage by Sap9 most often occurs in the vicinity of proximal basic amino acids, and in select cases also at basic and hydrophobic residues. Sap9 is found to cleave a large variety of proteins in a relatively short, similar to 1 h, period of time and it is efficient in a broad pH range, including slightly acidic, e. g., pH 5.5, conditions. Importantly, the resulting peptide mixtures contain representative peptides primarily in the target 3-7 kDa range. The utility and advantages of this enzyme in routine analysis of protein mixtures are demonstrated and the limitations are discussed. Overall, Sap9 has a potential to become an enzyme of choice in an extended bottom-up proteomics, which is technically ready to complement the traditional bottom-up proteomics for improved targeted protein structural analysis and expanded proteome coverage. Biological significance Advances in biological applications of mass spectrometry-based bottom-up proteomics are oftentimes limited by the extreme complexity of biological samples, e.g., proteomes or protein complexes. One of the reasons for it is in the complexity of the mixtures of enzymatically (most often using trypsin) produced short (

Elsevier Science Bv2014

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In-Spray Supercharging of Peptides and Proteins in Electrospray Ionization Mass Spectrometry

Luca Fornelli, Hubert Girault, Yu Lu, Sasa Miladinovic, Krzysztof Marek Piech, Yury Tsybin

Enhanced charging, or supercharging, of analytes in electrospray ionization mass spectrometry (ESI MS) facilitates high resolution MS by reducing an ion mass-to-charge (m/z) ratio, increasing tandem mass spectrometry (MS/MS) efficiency. ESI MS supercharging is usually achieved by adding a supercharging reagent to the electrospray solution. Addition of these supercharging reagents to the mobile phase in liquid chromatography (LC)-MS/MS increases the average charge of enzymatically derived peptides and improves peptide and protein identification in large-scale bottom-up proteomics applications but disrupts chromatographic separation. Here, we demonstrate the average charge state of selected peptides and proteins increases by introducing the supercharging reagents directly into the ESI Taylor cone (in-spray supercharging) using a dual-sprayer ESI microchip. The results are comparable to those obtained by the addition of supercharging reagents directly into the analyte solution or LC mobile phase. Therefore, supercharging reaction can be accomplished on a time-scale of ion liberation from a droplet in the ESI ion source.

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On the utility of predictive chromatography to complement mass spectrometry based intact protein identification

Hisham Ben Hamidane, Yury Tsybin

The amino acid sequence determines the individual protein three-dimensional structure and its functioning in an organism. Therefore, "reading" a protein sequence and determining its changes due to mutations or post-translational modifications is one of the objectives of proteomic experiments. The commonly utilized approach is gradient high-performance liquid chromatography (HPLC) in combination with tandem mass spectrometry. While serving as a way to simplify the protein mixture, the liquid chromatography may be an additional analytical tool providing complementary information about the protein structure. Previous attempts to develop "predictive" HPLC for large biomacromolecules were limited by empirically derived equations based purely on the adsorption mechanisms of the retention and applicable to relatively small polypeptide molecules. A mechanism of the large biomacromolecule retention in reversed-phase gradient HPLC was described recently in thermodynamics terms by the analytical model of liquid chromatography at critical conditions (BioLCCC). In this work, we applied the BioLCCC model to predict retention of the intact proteins as well as their large proteolytic peptides separated under different HPLC conditions. The specific aim of these proof-of-principle studies was to demonstrate the feasibility of using "predictive" HPLC as a complementary tool to support the analysis of identified intact proteins in top-down, middle-down, and/or targeted selected reaction monitoring (SRM)-based proteomic experiments.

Springer-Verlag2012

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