Biology subjects

Kruppa, G.

Publications and source records attributed to Kruppa, G..

4 recordsLinked to original sources

bioRxiv2023

The GlycoPaSER prototype as a real-time N-glycopeptide identification tool based on the PaSER parallel computing platform

Real-time database searching allows for simpler and automated proteomics workflows as it eliminates technical bottlenecks in high throughput experiments. Most importantly, it enables results dependent acquisition (RDA) where search results can be used to guide data acquisition during acquisition. This is especially beneficial for glycoproteomics since the wide range of physicochemical properties of glycopeptides lead to a wide range of optimal acquisition parameters. We established here the GlycoPaSER prototype by extending the Parallel Search Engine in Real-time (PaSER) functionality for real-time glycopeptide identification from fragmentation spectra. Glycopeptide fragmentation spectra were decomposed into peptide- and glycan-moiety spectra using common N-glycan fragments. Each moiety was subsequently identified by a specialized algorithm running in real-time. GlycoPaSER can keep up with the rate of data acquisition for real-time analysis with similar performance to other glycoproteomics software and produces results that are in line with literature reference data. The GlycoPaSER prototype presented here provides the first proof-of-concept for real-time glycopeptide identification that unlocks future development of RDA technology to transcend data acquisition.

bioinformatics↗

bioRxiv2022

Oxonium Ion-Guided Ion Mobility-Assisted Glycoproteomics on the timsTOF Pro

Spatial separation of ions in the gas-phase, providing information about their size as collisional cross-sections, can readily be achieved through ion mobility. The timsTOF Pro series combines a trapped ion mobility device with a quadrupole, collision-cell and a time-of-flight analyser to enable the analysis of ions at great speed. Here, we show that the timsTOF Pro is capable of physically separating N-glycopeptides from non-modified peptides and producing high-quality fragmentation spectra, both beneficial for glycoproteomics analyses of complex samples. The glycan moieties enlarge the size of glycopeptides compared to non-modified peptides, yielding a clear cluster in the mobilogram that, next to increased dynamic range from the physical separation of glycopeptides and non-modified peptides, can be used to make an effective selection filter for directing the mass spectrometer to analytes of interest. This new approach was applied to selected glycoproteins, human plasma- and neutrophil-derived glycopeptides. We show that the achieved physical separation, combined with the focussing of the mass spectrometer, allows for improved extraction of information from the samples, even at shorter LC gradients of 15 min. We validated our approach on human neutrophil and plasma samples of known make-up, in which we captured the anticipated glycan heterogeneity (paucimannose, phosphomannose, high mannose, hybrid and complex glycans) from plasma and neutrophil samples at the expected abundances. As the method is compatible with off-the-shelve data acquisition routines and data analysis software, it can readily be applied by any laboratory with a timsTOF Pro and is reproducible as demonstrated by a comparison between two laboratories.

biochemistry↗

bioRxiv2022

Highly multiplexed targeted plasma proteomics quantifies several hundred blood proteins in serum from colorectal carcinoma patients

The rapid analysis of human serum and plasma can provide deep insights into changes of the blood proteome in response to different patient treatments or diseases. Targeted proteomics techniques, like SRM and PRM, can be utilized to monitor proteins at high sensitivitym but so far were limited to smaller protein panels, which can be monitored in one experiment. The recently, on a Bruker tims-TOF pro mass spectrometer, developed parallel reaction monitoring-parallel accumulation - serial fragmentation (prm-PASEF) method expands the standard PRM method by using ion-mobility. The use of ion mobility as a fourth separation dimension increases the proteome coverage while reducing the length of the necessary chromatogeaphic separation. By combining an isotope-labeled reference standard, which covers 579 plasma proteins, we were able to quantify 565 proteins in plasma using prm-PASEF, with the least abundant protein being quantified at 7 amol. We continued the analysis by combining the isotype-labeled reference standard with dia-PASEF, which allowed the quantification of 549 proteins. Both methods were used to analyze 20 patient plasma samples from a colorectal cancer (CRC) cohort. The analysis identified 16 differentially regulated proteins between the CRC patient and control individual plasma samples. 15 of the 16 proteins showed a high correlation to the mRNA expression in CRC tumor samples, showing the technique s potential for the rapid identification of potential biomarkers in larger cohorts, abolishing the need for preselection of potential biomarker proteins. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/486663v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@eda5d4org.highwire.dtl.DTLVardef@220b01org.highwire.dtl.DTLVardef@1008757org.highwire.dtl.DTLVardef@1b56665_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Hydroxyl radical footprinting analysis of a human haptoglobin-hemoglobin complex

Methods of structural mass spectrometry have become more popular to study protein structure and dynamics. Among them, fast photochemical oxidation of proteins (FPOP) has several advantages such as irreversibility of modifications and more facile determination of the site of modification with single residue resolution. In the present study, FPOP analysis was applied to study the hemoglobin (Hb) - haptoglobin (Hp) complex allowing identification of respective regions altered upon the complex formation. Oxidative modifications were precisely localized on specific residues using a timsTOF Pro mass spectrometer. The data allowed determination of amino acids directly involved in Hb - Hp interactions and those located outside of the interaction interface yet affected by the complex formation. Data are available via ProteomeXchange with identifier PXD021621.

biochemistry↗