No abstract

Electron©detachment©dissociation©(EDD)©of©peptide©poly-anions©is©gentle©towards©post-translational© modifications© (PTMs)© and© produces© predictable© and© interpretable© fragment© ion types© (a·,© x ions).© However,© EDD© is© considered© an© inefficient© fragmentation© technique© and© has not© yet© been© implemented© in© large-scale© peptide© characterization© strategies.© We© successfully increased© the© EDD© fragmentation© efficiency© (up© to© 9%),© and© demonstrate© for© the© first© time© the utility©of©EDD-MS/MS©in©liquid©chromatography©time-scale©experiments.©Peptides©and phosphopeptides©were©analyzed©in©both©positive-©and©negative-ion©mode©using©electron capture/transfer© dissociation© (ECD/ETD)© and© EDD© in© comparison.© Using© approximately© 1 pmol©of©a©BSA©tryptic©digest,©LC-EDD-MS/MS©sequenced©14©peptides©(27%©aa©sequence coverage)© and© LC-ECD-MS/MS© sequenced© 19© peptides© (39%© aa© sequence© coverage).© Seven peptides© (18%© aa© sequence© coverage)© were© sequenced© by© both© EDD© and© ECD.© The© relative small©overlap©of©identified©BSA©peptides©demonstrates©the©complementarity©of©the©two dissociation© modes.© Phosphopeptide© mixtures© from© three© trypsin-digested© phosphoproteins were© subjected© to© LC-EDD-MS/MS© resulting© in© the© identification© of© five© phospho-peptides.© Of those,© one© was© not© found© in© a© previous© study© using© a© similar© sample© and© LC-ETD-MS/MS© in the© positive-ion© mode.© In© this© study,© the© ECD© fragmentation© efficiency© (15.7%© av.)© was© superior to© the© EDD© fragmentation© efficiency© (3.6%© av.).© However,© given© the© increase© in© amino© acid sequence© coverage© and© extended© PTM© characterization© the© new© regime© of© EDD© in© combination with© other© ion-electron© fragmentation© techniques© in© the© positive-ion© mode© is© a© step© towards© a more© comprehensive© strategy© of© analysis© in© proteome© research

SUMMARYManganese (Mn) constitutes an essential co-factor in the oxygen-evolving complex of photosystem II (PSII). Consequently, Mn deficiency reduces photosynthetic efficiency and leads to changes in PSII composition. In order to study these changes, multiplexed protein assays are advantageous. Here, we developed a multiplexed antibody-based assay and analysed selected PSII subunits in barley (Hordeum vulgare L.). A selection of antibodies were labelled with specific lanthanides and immunoreacted with thylakoids exposed to Mn deficiency after western blotting. Subsequently, western blot membranes were analysed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), which allowed selective and relative quantitative analysis via the different lanthanides. The method was evaluated against established liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) methods, based on data-dependent acquisition (DDA) and selected reaction monitoring (SRM). Manganese deficiency resulted in a general decrease in PSII protein abundances, an effect that was shown to be reversible upon Mn re-supplementation. Specifically, the extrinsic proteins PsbP and PsbQ showed Mn-dependent changes in abundances. Similar trends in the response to Mn deficiency at the protein level were observed when comparing DDA, SRM and LA-ICP-MS results. A biologically important exception to this trend was the loss of PsbO in the SRM analysis, which highlights the necessity of validating protein changes by more than one technique. The developed method enables a higher number of proteins to be multiplexed in comparison to existing immunoassays. Furthermore, multiplexed protein analysis by LA-ICP-MS provides an analytical platform with high throughput appropriate for screening large collections of plants.

Commercial feed fats, animal fat distillation residue, bleached earth oil and abused fryingfat were fractionated intomonomeric and dimeric triglycerides by means of gel permeation chromatograhy. Upon hydrolysis and methylation the mixtures were analysed by electron impact mass spectre metry using the integrated ion current technique. All samples contained a fraction of dimeric fatty acid methyl ester with molecular ions at m/z 584, 586,588 and 590, probably representing a great variety of both cyclic and linear structures.The individual dimers are not separated by this technique but the mass spectra of the multicomponent dimer fractions of all feed fats investigated showed remarkable resemblance. This may indicate that the dimeric fatty acids generated through intermoleculardimerization between two triglycerides are rather similar to those formed through intramolecular dimerization within the triglycerides. In two cases only trimeric fatty acids methyl esters were observed. The actual concentration of dimeric fatty acids was determined by HPLC-GPC to 3.7-13.70/0.

Phosphopeptide enrichment is an essential step in large-scale, quantitative phosphoproteomics studies by mass spectrometry. Several phosphopeptide affinity enrichment techniques exist, such as Immobilized Metal ion Affinity Chromatography (IMAC) and Metal Oxide Affinity Chromatography (MOAC). We compared Zirconium (IV) IMAC (Zr-IMAC) magnetic microparticles to more commonly used Titanium (IV) IMAC (Ti-IMAC) and TiO2 magnetic microparticles for phosphopeptide enrichment from simple and complex protein samples prior phosphopeptide sequencing and characterization by mass spectrometry (LC-MS/MS). We optimized sampleloading conditions to increase phosphopeptide recovery for Zr-IMAC, Ti-IMAC and TiO2 based workflows. The performance of Zr-IMAC was enhanced by 19-22% to recover up to 5173 phosphopeptides from 200 µg of protein extract from HepG2/C3A cells, making Zr-IMAC the preferred method for phosphopeptide enrichment in this study. Ti-IMAC and TiO2 performance were also optimized to improve phosphopeptide numbers by 28% and 35%, respectively. Furthermore, Zr-IMAC based phosphoproteomics in the magnetic microsphere format identified 23% more phosphopeptides than HPLC-based Fe(III)-IMAC for same sample amount (200 µg), thereby adding 37% more uniquely identified phosphopeptides. We conclude that Zr-IMAC improves phosphoproteome coverage and recommend that this affinity enrichment method should be more widely used in biological and biomedical studies of cell signalling and in the search for disease-biomarkers.