Biology subjects

Hober, A.

Publications and source records attributed to Hober, A..

4 recordsLinked to original sources

bioRxiv2023

Assessing the role of trypsin in quantitative plasma- and single-cell proteomics towards clinical application

Mass spectrometry-based bottom-up proteomics is rapidly evolving and routinely applied in large biomedical studies. Proteases are a central component of every bottom-up proteomics experiment, digesting proteins into peptides. Trypsin has been the most widely applied protease in proteomics, due to its characteristics. With ever-larger cohort sizes and possible future clinical application of mass spectrometry-based proteomics, the technical impact of trypsin becomes increasingly relevant. To assess possible biases introduced by trypsin digestion, we evaluated the impact of eight commercially available trypsins in a variety of bottom-up proteomics experiments, and across a range of protease concentrations and storage times. To investigate the universal impact of these technical attributes, we included bulk HeLa-cell lysate, human plasma and single HEK293 cells, which were analyzed over a range of Selected Reaction Monitoring (SRM), Data-Independent Acquisition (DIA), and Data-Dependent Acquisition (DDA) instrument methods on three LC-MS instruments. Quantification methods employed encompassed both label-free approaches and absolute quantification utilizing spike-in heavy-labeled recombinant protein fragment standards. Based on this extensive dataset, we report variations between commercial trypsins, their source, as well as their concentration. Furthermore, we provide suggestions on the handling of trypsin in large scale studies.

molecular biology↗

bioRxiv2021

Protein allocation and utilization in the versatile chemolithoautotroph Cupriavidus necator

Bacteria must balance the different needs for substrate assimilation, growth functions, and resilience in order to thrive in their environment. Of all cellular macromolecules, the bacterial proteome is by far the most important resource and its size is limited. Here, we investigated how the highly versatile knallgas bacterium Cupriavidus necator reallocates protein resources when grown on different limiting substrates and with different growth rates. We determined protein quantity by mass spectrometry and estimated enzyme utilization by resource balance analysis modeling. We found that C. necator invests a large fraction of its proteome in functions that are hardly utilized. Of the enzymes that are utilized, many are present in excess abundance. One prominent example is the strong expression of CBB cycle genes such as Rubisco during growth on fructose. Modeling and mutant competition experiments suggest that CO2-reassimilation through Rubisco does not provide a fitness benefit for heterotrophic growth, but is rather an investment in readiness for autotrophy. HighlightsO_LIA large fraction of the C. necator proteome is not utilized and not essential C_LIO_LIHighly utilized enzymes are more abundant and less variable C_LIO_LIAutotrophy related enzymes are largely underutilized C_LIO_LIRe-assimilation of CO2 via the CBB cycle is unlikely to provide a fitness benefit C_LI

systems biology↗

bioRxiv2019

Discovery of functional alternatively spliced PKM transcripts in human cancers

The association of pyruvate kinase muscle type (PKM) with survival of cancer patients is controversial. Here, we focus on different transcripts of PKM and investigate the association between their mRNA expression and the clinical survival of the patients in 25 different cancers. We find that the transcript encoding PKM2, and three other functional transcripts are prognostic in multiple cancers. Our integrative analysis shows that the functions of these four transcripts are highly conservative in different cancers. Next, we validate the prognostic effect of these transcripts in an independent kidney renal clear-cell carcinoma (KIRC) cohort and identify a prognostic signature which could distinguish high- and low-risk KIRC patients. Finally, we reveal the functional role of alternatively spliced PKM transcripts in KIRC, and discover the protein products of different transcripts of PKM. Our analysis demonstrated that alternatively spliced transcripts of not only PKM but also other genes should be considered in cancer studies, since it may enable the discovery and targeting of the right protein product for development of the efficient treatment strategies.

systems biology↗

bioRxiv2018

The human secretome - the proteins secreted from human cells

The proteins secreted by human tissues (the secretome) are important for the basic understanding of human biology, but also for identification of potential targets for future diagnosis and therapy. Here, we present an annotation of all predicted secreted proteins (n=2,623) with information about their spatial distribution in the human body. A high-throughput mammalian cell factory was established to create a resource of recombinant full-length proteins. This resource was used for phenotypic assays involving {beta}-cell dedifferentiation and for development of targeted proteomics assays. A comparison between host cells, including omics analysis, shows that many of the proteins that failed to be generated in CHO cells could be rescued in human HEK293 cells. In conclusion, the human secretome has been mapped and characterized to facilitate further exploration of the human secretome.

molecular biology↗