Jan Havliš

In-gel digestion for mass spectrometric characterization of proteins and proteomes

In-gel digestion of proteins isolated by gel electrophoresis is a cornerstone of mass spectrometry (MS)-driven proteomics. The 10-year-old recipe by Shevchenko et al. has been optimized to increase the speed and sensitivity of analysis. The protocol is for the in-gel digestion of both silver and Coomassie-stained protein spots or bands and can be followed by MALDI-MS or LC-MS/MS analysis to identify proteins at sensitivities better than a few femtomoles of protein starting material.

The Yeast APC/C Subunit Mnd2 Prevents Premature Sister Chromatid Separation Triggered by the Meiosis-Specific APC/C-Ama1

Cohesion established between sister chromatids during pre-meiotic DNA replication mediates two rounds of chromosome segregation. The first division is preceded by an extended prophase wherein homologous chromosomes undergo recombination. The persistence of cohesion during prophase is essential for recombination and both meiotic divisions. Here we show that Mnd2, a subunit of the anaphase-promoting complex (APC/C) from budding yeast, is essential to prevent premature destruction of cohesion in meiosis. During S- and prophase, Mnd2 prevents activation of the APC/C by a meiosis-specific activator called Ama1. In cells lacking Mnd2 the APC/C-Ama1 enzyme triggers degradation of Pds1, which causes premature sister chromatid separation due to unrestrained separase activity. In vitro, Mnd2 inhibits ubiquitination of Pds1 by APC/C-Ama1 but not by other APC/C holo-enzymes. We conclude that chromosome segregation in meiosis depends on the selective inhibition of a meiosis-specific form of the APC/C.

Swm1/Apc13 is an evolutionarily conserved subunit of the anaphase-promoting complex stabilizing the association of Cdc16 and Cdc27.

ABSTRACT The anaphase-promoting complex (APC/C) is a large ubiquitin-protein ligase which controls progression through anaphase by triggering the degradation of cell cycle regulators such as securin and B-type cyclins. The APC/C is an unusually complex ligase containing at least 10 different, evolutionarily conserved components. In contrast to APC/Cs role in cell cycle regulation little is known about the functions of individual subunits and how they might interact with each other. Here, we have analyzed Swm1/Apc13, a small subunit recently identified in the budding yeast complex. Database searches revealed proteins related to Swm1/Apc13 in various organisms including humans. Both the human and the fission yeast homologues are associated with APC/C subunits, and they complement the phenotype of an SWM1 deletion mutant of budding yeast. Swm1/Apc13 promotes the stable association with the APC/C of the essential subunits Cdc16 and Cdc27. Accordingly, Swm1/Apc13 is required for ubiquitin ligase activity in vitro and for the timely execution of APC/C-dependent cell cycle events in vivo.

Nucleocytoplasmic Shuttling of the Golgi Phosphatidylinositol 4-Kinase Pik1 Is Regulated by 14-3-3 Proteins and Coordinates Golgi Function with Cell Growth

The yeast phosphatidylinositol 4-kinase Pik1p is essential for proliferation, and it controls Golgi homeostasis and transport of newly synthesized proteins from this compartment. At the Golgi, phosphatidylinositol 4-phosphate recruits multiple cytosolic effectors involved in formation of post-Golgi transport vesicles. A second pool of catalytically active Pik1p localizes to the nucleus. The physiological significance and regulation of this dual localization of the lipid kinase remains unknown. Here, we show that Pik1p binds to the redundant 14-3-3 proteins Bmh1p and Bmh2p. We provide evidence that nucleocytoplasmic shuttling of Pik1p involves phosphorylation and that 14-3-3 proteins bind Pik1p in the cytoplasm. Nutrient deprivation results in relocation of Pik1p from the Golgi to the nucleus and increases the amount of Pik1p-14-3-3 complex, a process reversed upon restored nutrient supply. These data suggest a role of Pik1p nucleocytoplasmic shuttling in coordination of biosynthetic transport from the Golgi with nutrient signaling.

5-Methylcytosine as a marker for the monitoring of DNA methylation.

The extent of the DNA methylation of genomic DNA as well as the methylation pattern of many gene-regulatory areas are important aspects with regard to the state of genetic information, especially their expression. There is growing evidence that aberrant methylation is associated with many serious pathological consequences. As genetic research advances, many different approaches have been employed to determine the overall level of DNA methylation in a genome or to reveal the methylation state of particular nucleotide residues, starting from semiquantitative methods up to new and powerful techniques. In this paper, the currently employed techniques are reviewed both from the point of view of their relevance in genomic research and of their analytical application. The methods discussed include approaches based on chromatographic separation (thin-layer chromatography, high-performance liquid chromatography, affinity chromatography), separation in an electric field (capillary electrophoresis, gel electrophoresis in combination with methylation-sensitive restriction enzymes and/or specific sequencing protocols), and some other methodological procedures (mass spectrometry, methyl accepting capacity assay and immunoassays).

Evaluation of the possible proteomic application of trypsin from Streptomyces griseus

Trypsin (EC 3.4.21.4) is the protease of choice for proteome analysis using mass spectrometry of peptides in sample digests. In this work, trypsin from Streptomyces griseus (SGT) was purified to homogeneity from pronase. The enzyme was evaluated in in-gel digestion of protein standards followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) analyses of the digests. We recognized a remarkable cleavage performance of SGT. The number of produced and matching tryptic peptides was higher than in the case of commonly used bovine trypsin (BT) and allowed us to obtain higher identification scores in database searches. Interestingly, SGT was found to also generate nonspecific peptides whose sequencing by MALDI-TOF/TOF tandem mass spectrometry (MS/MS) revealed a partial F-X, Y-X, and W-X cleavage specificity. To suppress autolysis, either arginine or arginine plus lysine residues in SGT were modified by chemical reagents. In consequence, the autolytic pattern of SGT was reduced significantly, but specific activity dropped dramatically. As demonstrated by relative quantification of peptides at different times, SGT is more stable at 37 degrees C than is its bovine counterpart. We conclude that SGT represents a convenient alternative for proteomic applications involving protein digestion. Moreover, parallel digestions of sample aliquots by SGT and BT provide the possibility of combining partially different results (unique matching peptides) to improve protein identification.

Reactive dye ostazine black V-B: determination in the dye-bath and hydrolysis monitoring by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.

Monitoring the hydrolysis of Ostazine Black V-B reactive dye was found to be possible by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). All the desired substances were identified in a mixture of the dye-bath. The process of the substrate reactive dye hydrolysis was qualified with hydrolysis rate constants k(1) (2.0 +/- 0.3 h(-1)) and k(2) (0.6 +/- 0.1 h(-1)). The method developed is important for the dyeing industry and for following the colouration process.

Thermostable β-cyclodextrin conjugates of two similar plant amine oxidases and their properties

Syntheses of conjugates of garden pea (Pisum sativum) and grass pea (Lathyrus sativus) amine oxidases (PSAO and GPAO respectively) with BCD (β‐cyclodextrin), performed to improve the thermostability of the enzymes, are described in the present study. Periodate‐oxidized BCD reacted with the enzyme proteins via free primary amino groups in a buffered solution containing cyanoborohydride as a reductant. Although the specific activities of PSAO and GPAO partially decreased after modification, Km values determined for the best diamine substrates remained almost unchanged. Both the BCD conjugates could be incubated at 65 °C for 30 min without considerable inactivation, and the residual activity remained detectable even after incubation at 75 °C. The conjugates contained approx. 30% of neutral sugars. Molecular masses of BCD–PSAO and BCD–GPAO (180 kDa), as estimated by gel‐permeation chromatography, were higher compared with the value of 145 kDa for the native enzymes. This was in good correlation with the number of modified lysine residues determined by a spectrophotometric method. Peptide mass fingerprints of tryptic digests of BCD–PSAO and BCD–GPAO were less specific than those of the native enzymes when compared with the database sequence of PSAO. As a consequence of the modification, many unidentified peaks were observed in the digests of the studied conjugates that were not seen in the digests of native PSAO and GPAO. Only some of these peaks overlapped between BCD–PSAO and BCD–GPAO. The BCD conjugates described in the present study represent suitable candidates for biotechnological applications, e.g. in analyses using biosensors, which might benefit from increased storage stability and amine oxidation at high temperatures.

A simplified method for peptide de novo sequencing using 18 O labeling

Incorporation of an 18O atom into a peptide C-terminus by proteolytic cleavage in the presence of H218O is one of the most effective ways of enhancing tandem mass spectrometry (MS/MS)-based de novo sequencing. Incorporation is usually accomplished by procedures including vacuum-assisted drying of tryptic peptides extracted from gels, their subsequent reconstitution in a H216O/H218O mixture and re-treatment with trypsin. In the present work, we propose a simplified procedure for 18O incorporation into tryptic peptides by adding H218O and trypsin to the original digest solution. In comparison to published methods, the proposed protocol for peptide de novo sequencing brings significant advantages in analysis and workflow with no deterioration in method performance. We show that labeling by this simplified method leads to a highlighting of the y-ion fragment series in the peptide matrix-assisted laser desorption/ionization (MALDI)-MS/MS data, which facilitates MS/MS data interpretation. We also prove that eliminating acid extraction of peptides from gels does not result in a decrease in sequence coverage or a qualitative loss of particular peptides detectable by MALDI-MS. The method was examined by MALDI-MS/MS on bovine serum albumin and recombinant histidine kinase CKI1 from Arabidopsis thaliana, and was verified by de novo sequencing of tryptic peptides originating from Apodemus sylvaticus salivary proteins.

Terminology of bioanalytical methods (IUPAC Recommendations 2018)

Abstract Recommendations are given concerning the terminology of methods of bioanalytical chemistry. With respect to dynamic development particularly in the analysis and investigation of biomacromolecules, terms related to bioanalytical samples, enzymatic methods, immunoanalytical methods, methods used in genomics and nucleic acid analysis, proteomics, metabolomics, glycomics, lipidomics, and biomolecules interaction studies are introduced.