The great majority of colorectal cancers carry somatic mutations in one of two genes involved in the canonical Wnt/β-catenin signaling pathway: the adenomatous polyposis coli (APC) and β-catenin (CTNNB1) genes. Either type of genetic alteration results in accumulation of β-catenin and mimics the activation of Wnt signaling. Numerous attempts have therefore been made to develop therapeutics targeting the Wnt/β-catenin pathway. In colorectal cancer, however, due to the genetic inactivation of APC, only the molecules downstream of APC can be considered as therapeutic targets. We have therefore been searching for druggable target molecules downstream of APC, especially in the nucleus. We examined nuclear proteins of colorectal cancer cell lines immunoprecipitated with anti-TCF-4 or anti-β-catenin antibody by mass spectrometry and identified Traf2- and Nck-interacting kinase (TNIK) as an essential regulator of the TCF-4 or anti-β-catenin transcriptional complex. TNIK phosphorylated the conserved serine 154 residue of TCF4. Small interfering RNA (siRNA) targeting TNIK inhibited the proliferation of colorectal cancer cells and the growth of tumors produced by injecting colorectal cancer cells subcutaneously into immunodeficient mice. Several ATP-competing kinase inhibitors have been applied to cancer treatment and have shown significant activity. TNIK regulates Wnt signaling in the most downstream part of the pathway, and its inhibition is expected to block the signal even in colorectal cancer cells with APC gene mutation. TNIK is considered to be a feasible target of pharmacological intervention for manipulation of the aberrant Wnt signaling pathway in colorectal cancer.

The most successful clinical application of proteomics to date is MALDI-TOF MS based bacterial identification. As compared with the conventional methods, the MS-based method is more quick and simple; indeed, there has been a revolutionary shift in clinical diagnostic microbiology.

A small portion of a cultured bacterial colony is directly subjected to MALDI-TOF MS. The final mass spectral signature is composed of peaks originating from bacterial proteins including mainly ribosomal proteins. The spectral profiles are compared with a library of known spectra and a result is generated within 10 min instead of almost one day when conducted by the traditional methods.

The rate of successful identification at the species levels, however, is still not 100% for various reasons such as incomplete databases. We have found that in-house refinement of commercial database by incorporating MS spectra of clinical isolates obtained locally can significantly improve the identification rate. Also, for some pathogens, extensive pretreatment of the samples is mandatory to obtain appropriate proteomic profiles. Progress has been made for direct analysis of bacteria in urine, cerebrospinal fluid (CSF), and blood. Beyond identification of bacteria, MS is increasingly used for detection of antibiotic resistance. For this purpose, LC/MS/MS may play roles as well.

To meet with rapid progress in clinical applications of MS, the Japanese Society for Biomedical Mass Spectrometry has started to certify medical mass spectrometrist since 2013. As of June 2017, a total of 298 have been certified.

Post-translational modifications extend the varieties of chemical characteristics of fundamental 20 amino acid residues, and thereby enable proteins to function in diverse biological processes. Of the post-translational modifications, lysine acylations change the chemical properties of lysine residues through the transfer of acyl groups with the different charge states and bulkiness. Recent development of mass spectrometric technology discovered several lysine acylations. Furthermore, these acylations were identified on hundreds to thousands of proteins, suggesting complex system involved by lysine acylations. To understand fundamental and common functions by lysine acylations among organisms ranging from bacteria to human, we characterized lysine acylations such as acetylation, propionylation, and succinylation in bacteria. Here, we mainly described our studies on lysine propionylation and succinylation. We identified these lysine acylations on a proteome-wide scale in 5 bacterial species. Furthermore, by using protein tertiary structure information, we speculated the roles of dozens of lysine acylations. Interestingly, many acylation sites were located at the positions near the bound ligands. Comparison of the characteristics of these two types of acylation suggested their different roles in cellular processes. To evaluate which modification effects are different between propionylation and succinylation, we conducted in silico docking simulation analysis of unmodified, propionylated, and succinylated enzymes with its ligand. The result suggested that lysine propionylation and succinylation can have a moderate and strong influence on protein function, respectively.

Chemically synthesized peptides labeled with stable isotopes are currently used as internal standards in mass spectrometry for protein quantitation. Although the use of inexpensive and crude peptides for the relative quantification of proteome dynamics has seen an increase in recent years, the synthesis of high quality internal standard peptides for measuring the absolute amount of a target protein remains expensive, making it difficult to achieve absolute protein quantification using synthetic peptides on a proteome-wide scale. Instead, a quantitative strategy using a peptide conjugate called QconCAT has been proposed as an alternative approach for absolute quantification of multiple target proteins; however, the establishment of a stable QconCAT biosynthesis system is required for its widespread use. This review focuses on the multiplexed co-synthesis approach for QconCATs using a wheat germ cell-free system with a robust translation machinery. Ultra-throughput QconCAT production in the cell-free system allows rapid construction of large-scale standard peptide libraries and accelerates attempts to quantify global proteome dynamics in various biological processes.