Biology subjects

Olsen, J. V.

Publications and source records attributed to Olsen, J. V..

4 recordsLinked to original sources

bioRxiv2018

Protein aggregation capture on microparticles enables multi-purpose proteomics sample preparation

Universal proteomics sample preparation is challenging due to the high heterogeneity of biological samples. Here we describe a novel mechanism that exploits the inherent instability of denatured proteins for non-specific immobilization on microparticles by protein aggregation capture. To demonstrate the general applicability of this mechanism, we analyzed phosphoproteomes, tissue proteomes, and interaction proteomes as well as dilute secretomes. The findings presents a practical, sensitive and cost-effective proteomics sample preparation method.

biochemistry↗

bioRxiv2018

Alternative translation initiation generates a functionally distinct isoform of the stress-activated kinase MK2

Shaping of the proteome by alternative translation is an important mechanism of post-transcriptional gene regulation. It can lead to the expression of multiple protein isoforms originating from the same mRNA. Here we show that a novel, abundant and long isoform of the stress/p38MAPK-activated kinase MK2, a key regulator of transcription, migration, death signaling and post-transcriptional gene regulation, is constitutively translated from an alternative CUG translation initiation start site located in the 5'UTR of its mRNA. GC-rich sequences and putative G-quadruplex structures influence the usage of that codon as a translation initiation start site and the RNA helicase eIF4A1 is needed to ensure alternative isoform translation. We recapitulated the usage of the alternative start codon and determined the molecular properties of the short and a long MK2 isoforms. Phenotypically, only the short isoform phosphorylated Hsp27, supported migration and stress-induced immediate early gene (IEG) expression. Interaction profiling by quantitative mass-spectrometry revealed short isoform-specific binding partners that were associated with migration. In contrast, the long isoform contains additional putative phosphorylation sites in its unique N-terminus. In sum, our data reveal a longer and previously non-described isoform of MK2 with distinct physiological properties originating from alternative translation.

molecular biology↗

bioRxiv2018

Limits for resolving tandem mass tag reporter ions with identical integer mass using phase constrained spectrum deconvolution

A popular method for peptide quantification relies on isobaric labeling such as tandem mass tags (TMT) which enables multiplexed proteome analyses. Quantification is achieved by reporter ions generated by fragmentation in a tandem mass spectrometer. However, with higher degrees of multiplexing, the smaller mass differences between the reporter ions increase the mass resolving power requirements. This contrasts with faster peptide sequencing capabilities enabled by lowered mass resolution on Orbitrap instruments. It is therefore important to determine the mass resolution limits for highly multiplexed quantification when maximizing proteome depth. Here we defined the lower boundaries for resolving TMT reporter ions with 0.0063 Da mass differences using an ultra-high-field Orbitrap mass spectrometer. We found the optimal method depends on the relative ratio between closely spaced reporter ions and that 64 ms transient acquisition time provided sufficient resolving power for separating TMT reporter ions with absolute ratio changes up to 16-fold. Furthermore, a 32 ms transient processed with phase-constrained spectrum deconvolution provides >50% more identifications with >99% quantified, but with a slight loss in quantification precision and accuracy. These findings should guide decisions on what Orbitrap resolution settings to use in future proteomics experiments relying on TMT reporter ion quantification with identical integer masses.

biochemistry↗

bioRxiv2018

A novel LC system embeds analytes in pre-formed gradients for rapid, ultra-robust proteomics

To further integrate mass spectrometry (MS)-based proteomics into biomedical research and especially into clinical settings, high throughput and robustness are essential requirements. They are largely met in high-flow rate chromatographic systems for small molecules but these are not sufficiently sensitive for proteomics applications. Here we describe a new concept that delivers on these requirements while maintaining the sensitivity of current nano-flow LC systems. Low-pressure pumps elute the sample from a disposable trap column, simultaneously forming a chromatographic gradient that is stored in a long storage loop. An auxiliary gradient creates an offset, ensuring the re-focusing of the peptides before the separation on the analytical column by a single high-pressure pump. This simplified design enables robust operation over thousands of sample injections. Furthermore, the steps between injections are performed in parallel, reducing overhead time to a few minutes and allowing analysis of more than 200 samples per day. From fractionated HeLa cell lysates, deep proteomes covering more than 130,000 sequence unique peptides and close to 10,000 proteins were rapidly acquired. Using this data as a library, we demonstrate quantitation of 5200 proteins in only 21 min. Thus, the new system-termed Evosep One-analyzes samples in an extremely robust and high throughput manner, without sacrificing in depth proteomics coverage.

biochemistry↗