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High-performance liquid chromatography (HPLC) is widely used for separation of complex peptide mixtures before mass spectrometry-based proteome analysis. In this analysis, reversed phase HPLC (RPHPLC) using non-polar stationary phases such as surface-modified silica containing alkyl groups (e.g., C18) is typically employed. Because of the high heterogeneity of proteomic samples, multidimensional separation approaches gained increasing attention recently to tackle this complexity and extremely high range of concentrations. In two-dimensional liquid chromatography, hydrophilic interaction chromatography (HILIC) is often a method of choice for combination with RP-HPLC because it uses reversed-phase type eluents and allows efficient separation of polar peptides. Due to the high degree of orthogonality in this two-dimensional separation space, it is tempting to develop approaches for predicting peptide retention times for HILIC-based separations similar to the ones for RP-HPLC. Recent successful efforts in this area were focused on developing retention coefficient (RC)-based approaches. Herein, we explored the feasibility of using a statistical thermodynamic model for prediction of peptide retention times in HILIC separations and determined the phenomenological parameters of the model for a bare silica column. The performance of the developed model was tested using HPLC-MS analysis of a set of synthetic peptides, as well as a tryptic peptide mixture. Keywords: HILIC, peptide separation, proteomics, retention time prediction DOI: 10.1134/S1061934817140076, INTRODUCTION Proteomics is a general term for a variety of approaches and techniques used for large-scale identification and quantification of proteins present in certain cell, tissue, or body fluid of [...]

Morpheus is a search algorithm developed recently for high-resolution tandem mass spectra. According to the developers, its intrinsic property is discriminating short sequence length peptides. Therefore, elimination of direct comparisons between peptide spectrum matches (PSMs) for short and long peptides may potentially increase the search sensitivity for a given FDR level. In the proposed approach, all PSMs are grouped according to the number of matched fragment ions, followed by separate filtering of identifications in each group using target-decoy approach. The approach is applied to Morpheus output results and does not cause a significant increase in the overall data analysis time. The proposed approach was implemented as a Python command-line tool, called GroupFilter. Several data sets from different types of mass spectrometers were used for testing of the software, including the data from the original Morpheus search engine paper. Separate FDR filtering for grouped identifications increased the number of identified peptides by up to 18% compared with the default Morpheus post-processing procedure. The proposed approach can be considered as an addition to the Morpheus search engine. Keywords: proteomics, tandem mass spectrometry, peptide identification, Morpheus, post-search processing DOI: 10.1134/S1061934816130074, INTRODUCTION Currently, a variety of search algorithms for protein identification in shotgun proteomics have been developed [1-8]. Comparison of the efficiency of different search engines has shown that the results [...]

The effect of parameters of a binary mobile phase (chemical properties of components, gradient profile, flow rate, and pH) on the separation of peptides and proteins is theoretically investigated using a model of critical chromatography (model BioLCCC). It is demonstrated that the retention volume of peptides can be inverted by changing separation conditions. Keywords: high performance liquid chromatography, proteins and peptides, critical chromatography of biopolymers, The BioLCCC model of critical chromatography satisfactorily describes the nature of the macromolecules separation with different sequences, including peptides and proteins [1-8]. This model combines both microscopic parameters, such as [...]

High-throughput proteomics technologies are gaining popularity in different areas of life sciences. One of the main objectives of proteomics is characterization of the proteins in biological samples using liquid chromatography/mass spectrometry analysis of the corresponding proteolytic peptide mixtures. Both the complexity and the scale of experimental data obtained even from a single experimental run require specialized bioinformatic tools for automated data mining. One of the most important tools is a so-called proteomics search engine used for identification of proteins present in a sample by comparing experimental and theoretical tandem mass spectra. The latter are generated for the proteolytic peptides derived from a protein database. Peptide identifications obtained with the search engine are then scored according to the probability of a correct peptide-spectrum match. The purpose of this work was to perform a comparison of different search algorithms using data acquired for complex protein mixtures, including both annotated protein standards and clinical samples. The comparison was performed for three popular search engines: commercially available Mascot, as well as open-source X!Tandem and OMSSA. It was shown that the search engine OMSSA identifies in general a smaller number of proteins, while X!Tandem and Mascot deliver similar performance. We found no compelling reasons for using the commercial search engine instead of its open source competitor. Keywords: proteomics, peptide identification, tandem mass spectrometry, search algorithms DOI: 10.1134/S1061934815140075, INTRODUCTION A significant amount of biological research focuses on proteomics, which analyzes proteins in cells or tissues, as well as their interactions and modifications. Liquid chromatography/mass spectrometry (LC-MS) has become [...]

Inversion of the order of peptide elution in reversed-phase liquid chromatography under changing separation conditions, such as gradient slope has been considered. Using a six- protein proteolytic peptide standard and available literature data, the occurrence frequency and importance of this phenomenon in proteomic studies utilizing methods of shotgun proteomics and accurate mass and time tags have been evaluated. Feasibility of qualitative and quantitative description of peptide elution order inversion has been demonstrated using a model of critical liquid chromatography. Existing approaches to predict peptide separation and directions of the shifts of chromatographic peaks when the gradient profile changes have been compared. Keywords: proteomics, mass spectrometry, inversion of chromatographic elution orders of peptides, accurate mass and retention time tags, critical liquid chromatography DOI: 10.1134/S1061934812130102, INTRODUCTION Inversion of peptide elution order in reversed-phase liquid chromatography under changing separation parameters, e.g. solvent flow rate, column size and gradient profile, has been widely discussed in literature since [...]

Inversion of the order of peptide elution in reversed-phase liquid chromatography under changing separation conditions, such as gradient slope has been considered. Using a six-protein proteolytic peptide standard and available literature data, the occurrence frequency and importance of this phenomenon in proteomic studies utilizing methods of shotgun proteomics and accurate mass and time tags have been evaluated. Feasibility of qualitative and quantitative description of peptide elution order inversion has been demonstrated using a model of critical liquid chromatography. Existing approaches to predict peptide separation and directions of the shifts of chromatographic peaks when the gradient profile changes have been compared. Keywords: proteomics, mass spectrometry, inversion of chromatographic elution orders of peptides, accurate mass and retention time tags, critical liquid chromatography DOI: 10.1134/S1061934812130102, INTRODUCTION Inversion of peptide elution order in reversed-phase liquid chromatography under changing separation parameters, e.g. solvent flow rate, column size and gradient profile, has been widely discussed in literature since [...]

An atmospheric pressure ionization source based on desorption electrospray ionization technology for a bench-top hybrid FTICR mass spectrometer is described. The ion source was characterized using low-molecular-weight-weight pharmaceutical samples. The dependences of signal intensities on various experimental parameters (solvent composition, surface temperature, spray voltage, etc.) were studied. Based on the results obtained, plausible mechanisms of desorption electrospray ionization for the analytes under the study are discussed. Keywords: mass spectrometry, atmospheric pressure ionization sources, desorption electrospray ionization. DOI: 10.1134/S1061934811140024, INTRODUCTION Nowadays mass spectrometry occupies the leading position among the methods of rapid and continuous analysis of the ecological state of the environment; it is widely used in criminology and [...]

Shotgun proteomics [1], which is based on liquid chromatography--mass spectrometry (LC--MS), is the most commonly employed technique for qualitative and quantitative analysis of protein samples. In the so-called bottom-up approach, [...]