Metastasis-associated C.,4A, A GPI-Anchored protein cleaved by Adam10 and Adam 17

Comunicaciones a congreso

Metastasis-associated C4. 4A, A GPI-anchored protein cleaved by ADAM10 and ADAM17

Comunicaciones a congreso

The Impact II, a Very High-Resolution Quadrupole Time-of-Flight Instrument (QTOF) for Deep Shotgun Proteomics

Hybrid quadrupole time-of-flight (QTOF) mass spectrometry is one of the two major principles used in proteomics. Although based on simple fundamentals, it has over the last decades greatly evolved in terms of achievable resolution, mass accuracy, and dynamic range. The Bruker impact platform of QTOF instruments takes advantage of these developments and here we develop and evaluate the impact II for shotgun proteomics applications. Adaption of our heated liquid chromatography system achieved very narrow peptide elution peaks. The impact II is equipped with a new collision cell with both axial and radial ion ejection, more than doubling ion extraction at high tandem MS frequencies. The new reflectron and detector improve resolving power compared with the previous model up to 80%, i.e. to 40,000 at m/z 1222. We analyzed the ion current from the inlet capillary and found very high transmission (>80%) up to the collision cell. Simulation and measurement indicated 60% transfer into the flight tube. We adapted MaxQuant for QTOF data, improving absolute average mass deviations to better than 1.45 ppm. More than 4800 proteins can be identified in a single run of HeLa digest in a 90 min gradient. The workflow achieved high technical reproducibility (R2 > 0.99) and accurate fold change determination in spike-in experiments in complex mixtures. Using label-free quantification we rapidly quantified haploid against diploid yeast and characterized overall proteome differences in mouse cell lines originating from different tissues. Finally, after high pH reversed-phase fractionation we identified 9515 proteins in a triplicate measurement of HeLa peptide mixture and 11,257 proteins in single measurements of cerebellum-the highest proteome coverage reported with a QTOF instrument so far

Susceptibility to tuberculosis is associated with variants in the ASAP1 gene encoding a regulator of dendritic cell migration

Human genetic factors predispose to tuberculosis (TB). We studied 7.6 million genetic variants in 5,530 people with pulmonary TB and in 5,607 healthy controls. In the combined analysis of these subjects and the follow-up cohort (15,087 TB patients and controls altogether), we found an association between TB and variants located in introns of the ASAP1 gene on chromosome 8q24 (P = 2.6 × 10−11 for rs4733781; P = 1.0 × 10−10 for rs10956514). Dendritic cells (DCs) showed high ASAP1 expression that was reduced after Mycobacterium tuberculosis infection, and rs10956514 was associated with the level of reduction of ASAP1 expression. The ASAP1 protein is involved in actin and membrane remodeling and has been associated with podosomes. The ASAP1-depleted DCs showed impaired matrix degradation and migration. Therefore, genetically determined excessive reduction of ASAP1 expression in M. tuberculosis–infected DCs may lead to their impaired migration, suggesting a potential mechanism of predisposition to TB

Protein mixtures using two-dimensional chromatography and ion trap MS

Studying proteomes is of central interest in life sciences, because expression and degradation of proteins are highly dynamic processes, which admit cells to adapt to changing conditions like drugs, temperature changes etc. In this respect, e.g. the discovery of proteins with unknown functionality or the investigation o fmetabolic pathways are main topics

MALDI-TOF-MS integrated workflow for food authenticity investigations: An untargeted protein-based approach for rapid detection of PDO feta cheese adulteration

Advances in Matrix-assisted Laser Desorption/Ionization -Time-Of-Flight Mass Spectrometry (MALDI-TOF-MS) have led to its supremacy for complex assessment of food authenticity studies, like dairy products fraud, holding promise for the discovery of potential authenticity (bio)markers. In this study, an integrated untargeted protein-based workflow in combination with advanced chemometrics is presented, to address authenticity challenges in PDO feta cheese which is legally manufactured by the mixture of sheep/goat milk. Potential markers attributed to specific animal origin were found from protein profiles acquired for authentic feta and white cheeses (prepared from cow milk), belonging to 4 kDa–18.5 kDa mass area. Rapid detection of feta cheese adulteration from cow milk was also achieved down to 1% adulteration level. The discriminative models showed high predictive ability for feta cheese authenticity (Q2 = 0.920, RMSEE = 0.053) and its adulteration (Q2 = 0.835, RMSEE = 0.121), introducing a reliable approach in routine analysis. The methodology was successfully applied in detection of cow milk in sheep yoghurt. © 2021 Elsevier Lt