Dataset Information

Analysis of FucT and XylT1B mutants in C. reinhardtii

ABSTRACT: Secreted proteins found in the supernatant of different mutants were analyzed by mass spectrometry employing In source-CID. After analysis of intact N-glycopeptides on MS1 level, N-glycan compositions found were compared between two strains, consequently unraveling the molecular function of enzymes knocked down. Hereby, the role of a putative fucosyltransferase and the identity of a second core- xylosyltransferase could be confirmed.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Chlamydomonas Reinhardtii

TISSUE(S): Culture Supernatant, Photosynthetic Cell

SUBMITTER: Anne Oltmanns

LAB HEAD: Michael Hippler

PROVIDER: PXD012107 | Pride | 2019-11-19

REPOSITORIES: Pride

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Project description:Formaldehyde fixation is widely used for long-term maintenance of tissue. However, due to formaldehyde-induced cross-links, fixated tissue proteins are difficult to extract hampering the performance of mass spectrometry (MS)-proteomic analysis on formaldehyde-fixated tissue. Recent years saw the use of different combinations of high-temperature and solubilizing agents (usually derived from antigen retrieval techniques) to unravel formaldehyde-fixated paraffin-embedded tissue proteomes. However, in order to achieve protein extraction yields similar to fresh-frozen tissue high-temperature heating is necessary. Such harsh extraction conditions may affect, labile post-translational modifications such as phosphorylations resulting in the loss of important protein information. The objective of the present work is to assess cleavable fixative reagents that allow tissue preservation as well as efficient protein extraction from fixated tissue for MS-proteomics under mild conditions. To this regard, we investigated disuccinimidyl tartrate (DST) and dithiobis[succinimidylpropionate] (DSP) as cleavable fixating reagents. These compounds crosslink proteins by reacting with amino groups leading to amide bond formation. Linkers can be cleaved with sodium metaperiodate (cis-diols) or reducing agents (disulfide bonds), respectively. Our results show that reversible protein crosslinking allows tissue fixation with morphology preservation comparable to formalin. In addition, cleavage of DSP improves protein recovery from fixated tissue by a factor of 18 and increases the number of identified proteins by approximately 20% under mild extraction conditions avoiding the need for sample boiling, which could affect labile post-translational modifications. A major advantage of DSP over formaldehyde is the introduction of well-defined protein modifications that can be taken into account during database searching. In contrast to DSP fixation, DST fixation followed by periodic cleavage, while effective, resulted in side reactions that prevented effective protein extraction and subsequent protein identification. Protein crosslinkers, which can be cleaved under mild conditions, are thus viable alternatives to formaldehyde as tissue fixatives facilitating protein analysis from paraffin embedded, fixated tissue.

Dataset Information

Analysis of FucT and XylT1B mutants in C. reinhardtii

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