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An enzymatic approach to label peptide N-termini with isotope-coded affinity tags is presented. This method exploits the high activity of trypsin for peptide synthesis in organic solvents. A cosubstrate containing a stable isotope-coded Arg residue and a biotin tag was synthesized. When the cosubstrate was incubated with tryptic peptides and trypsin in ethanol solution, the stable isotope-coded affinity tag was specifically coupled onto the N-termini of peptides via the formation of new peptide bonds. The labeled peptides were specifically enriched by avidin affinity chromatography and then were submitted to liquid chromatography-tandem mass spectrometry (LC/MS/MS) for quantification. This enrichment step effectively reduced the interference by unlabeled peptides. The excellent performance of this approach was demonstrated by labeling standard peptides as well as a mouse liver digest. In addition to one amino acid residue, a few dipeptide tags were also introduced to the N-termini of peptides successfully by this enzymatic approach. It was found that the identifications for samples labeled with these tags were highly complementary. Coupling a short sequence tag onto peptides could be an effective approach to improve the coverage for proteome analysis. [ABSTRACT FROM AUTHOR]

Conventional sample preparation protocols for phosphoproteome analysis require multiple time-consuming and labor-intensive steps, including cell lysis, protein extraction, protein digestion, and phosphopeptide enrichment. In this study, we found that the presence of a large amount of trypsin in the sample did not interfere with phosphopeptide enrichment and subsequent LC-MS/MS analysis. Taking advantage of fast digestion achieved with high trypsin-to-protein ratio, we developed a novel concurrent lysis-digestion method for phosphoproteome analysis. In this method, the harvested cells were first placed in a lysis buffer containing a huge amount of trypsin. After ultrasonication, the cells were lysed and the proteins were efficiently digested into peptides within one step. Thereafter, tryptic digest was subjected to phosphopeptide enrichment, in which unphosphorylated peptides, trypsin, and other components incompatible with LC-MS/MS analysis were removed. Compared with conventional methods, better phosphoproteome coverage was achieved in this new one-step method. Because protein solubilization and cell lysis were facilitated by fast protein digestion, the complete transformation of cell pellets into the peptide mixture could be finished within 25 min, while it would take at least 16 h for conventional methods. Hence, our method, which integrated cell lysis, protein extraction, and protein digestion into one step, is rapid and convenient It is expected to have broad applications in phosphoproteomics analysis. [ABSTRACT FROM AUTHOR]

A problem for "shot-gun" proteomics is that the peptides generated in the proteolysis step overwhelm the analytical capacity of current LC-MS/MS systems. A straightforward approach to overcome this problem is to reduce the sample complexity by isolating the representative peptides of each protein. In this study, we presented a facile solid-phase capture-release approach to selectively enrich the peptides with N-terminal serine/threonine from protein digests. This method exploited the highly efficient reaction between an aldehyde group and a hydrazine group. The excellent performance of this approach was validated using synthetic peptides as well as complex protein digests. It was found that high enrichment specificity could be obtained and the identifications for complex samples with and without enrichment were highly complementary. Besides, the enrichment of peptides with serine/threonine adjacent to different protease cleavage sites demonstrated that our method was able to enrich peptides from protein digests in a sequence specific way. As a result, this new approach provides a simple way to reduce sample complexity and facilitates the identification of low-abundance proteins. [ABSTRACT FROM AUTHOR]

Due to its constitutive activity and ubiquitous distribution, CK2 is the most pleiotropic kinase among the individual members of the protein kinase superfamily. Identification of CK2 substrates is vital to decipher its role in biological processes. However, only a limited number of CK2 substrates were identified so far. In this study, we developed an integrated phosphoproteomics workflow to identify the CK2 substrates in large scale. First, in vitro kinase reactions with immobilized proteomes were combined with quantitative phosphoproteomics to identify in vitro CK2 phosphorylation sites, which leaded to identification of 988 sites from 581 protein substrates. To reduce false positives, we proposed an approach by comparing these in vitro sites with the public databases that collect in vivo phosphorylation sites. After the removal of the sites that were excluded in the databases, 605 high confident CK2 sites corresponding to 356 proteins were retained. The CK2 substrates identified in this study were based on the discovery mode, in which an unbiased overview of CK2 substrates was provided. Our result revealed that CK2 substrates were significantly enriched in the spliceosomal proteins, indicating CK2 might regulate the functions of spliceosome. [ABSTRACT FROM AUTHOR]

Efficient killing of nematodes by Stropharia rugosoannulata Farlow ex Murrill cultures was observed. This fungus showed the ability to immobilize the free-living nematode Panagrellus redivivus Goodey within minutes and to immobilize the pine wilt nematode Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle within hours on agar plates. Moreover, P. redivivus worms were completely degraded by the fungus within 24 to 48 h. The cultures of S. rugosoannulata studied shared the characteristic of abundantly producing cells with finger-like projections called acanthocytes. We showed that the nematode-attacking activity of this fungus is carried out by these spiny acanthocytes and that mechanical force is an important factor in the process. Furthermore, the growth and nematode-attacking activity of the fungus in soil were also determined, and our results suggest that acanthocytes are functional in soil. [ABSTRACT FROM AUTHOR]