Advances in MALDI mass spectrometry in clinical diagnostic applications.

The concept of matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) was first reported in 1985. Since then, MALDI MS technologies have been evolving, and successfully used in genome, proteome, metabolome, and clinical diagnostic research. These technologies are high-throughput and sensitive. Emerging evidence has shown that they are not only useful in qualitative and quantitative analyses of proteins, but also of other types of biomolecules, such as DNA, glycans, and metabolites. Recently, parallel fragmentation monitoring (PFM), which is a method comparable to selected reaction monitoring, has been reported. This highlights the potentials of MALDI-TOF/TOF tandem MS in quantification of metabolites. Here we critically review the applications of the major MALDI MS technologies, including MALDI-TOF MS, MALDI-TOF/TOF MS, SALDI-TOF MS, MALDI-QqQ MS, and SELDI-TOF MS, to the discovery and quantification of disease biomarkers in biological specimens, especially those in plasma/serum specimens. Using SELDI-TOF MS as an example, the presence of systemic bias in biomarker discovery studies employing MALDI-TOF MS and its possible solutions are also discussed in this chapter. The concepts of MALDI, SALDI, SELDI, and PFM are complementary to each other. Theoretically, all these technologies can be combined, leading to the next generation of the MALDI MS technologies. Real applications of MALDI MS technologies in clinical diagnostics should be forthcoming.

Clathrin light chain A facilitates small extracellular vesicle uptake to promote hepatocellular carcinoma progression.

BACKGROUND: Endocytosis is a fundamental process for internalizing small extracellular vesicles (sEVs). The present study aimed to elucidate the role of clathrin light chain A (CLTA) in sEV uptake in hepatocellular carcinoma (HCC). MATERIALS AND METHODS: CLTA expression was analyzed by bioinformatics, quantitative PCR and immunohistochemistry. The clinical relevance of CLTA was analyzed by Fisher's exact test, Kaplan-Meier analysis, and multivariate cox regression model. The functions of CLTA in sEV uptake and cancerous properties were examined by PKH67-sEV uptake, MTT, colony formation, and transwell assays. Mass spectrometry was used to identify the downstream effectors of CLTA. CLTA inhibitor, Pitstop 2, was tested in a mouse model of patient-derived xenografts (PDXs). RESULTS: CLTA expression was higher in tumor tissues than in non-tumorous liver tissues and progressively increased from the early to late tumor stage. CLTA overexpression was associated with larger tumor size and poor prognosis in HCC. Cellular CLTA contributed to the sEV uptake, resulting in enhanced cancerous properties. Mechanistically, CLTA increases capping actin protein gelsolin-like (CAPG) expression to facilitate sEV uptake, thereby promoting the proliferation, motility, and invasiveness of HCC cells. What's more, the CLTA inhibitor Pitstop 2 alone or in combination with sorafenib attenuated tumor growth in mice implanted with PDXs. CONCLUSIONS: The study reveals the role of CLTA in sEV uptake to promote HCC progression. Inhibition of CLTA and its mediated pathway illuminate a new therapeutic strategy for HCC patients.

A magnetic bead-based serum proteomic fingerprinting method for parallel analytical analysis and micropreparative purification.

ProteinChip surface-enhanced laser desorption/ionization technology and magnetic beads-based ClinProt system are commonly used for semi-quantitative profiling of plasma proteome in biomarker discovery. Unfortunately, the proteins/peptides detected by MS are non-recoverable. To obtain the protein identity of a MS peak, additional time-consuming and material-consuming purification steps have to be done. In this study, we developed a magnetic beads-based proteomic fingerprinting method that allowed semi-quantitative proteomic profiling and micropreparative purification of the profiled proteins in parallel. The use of different chromatographic magnetic beads allowed us to obtain different proteomic profiles, which were comparable to those obtained by the ProteinChip surface-enhanced laser desorption/ionization technology. Our assays were semi-quantitative. The normalized peak intensity was proportional to concentration measured by immunoassay. Both intra-assay and inter-assay coefficients of variation of the normalized peak intensities were in the range of 4-30%. Our method only required 2 microL of serum or plasma for generating enough proteins for semi-quantitative profiling by MALDI-TOF-MS as well as for gel electrophoresis and subsequent protein identification. The protein peaks and corresponding gel spots could be easily matched by comparing their intensities and masses. Because of its high efficiency and reproducibility, our method has great potentials in clinical research, especially in biomarker discovery.

Chemical and pharmacological evaluations on the extract of Scutellaria baicalensis Georgi (Huang-Qin) prepared by various extraction methods.

BACKGROUND: This study reported a comprehensive approach (comparing the extraction yields, chemical profiles, antioxidant properties and CYP450-inhibitory effects) to evaluated the effectiveness of various extraction methods [microwave-assisted extraction using water (MAE-W), heat reflux extraction using water (HRE-W), ultrasonic extraction using 70 % ethanol and ultrasonic extraction using ethanol (UE-E)] for Huang-Qin (HQ), the dried root of Scutellaria baicalensis Georgi. RESULTS: The HQ extraction efficiency by MAE-W was the best. The chemical profiles of extracts obtained using HRE-W and MAE-W were similar; whereas more flavones but less flavone glycosides were detected in the UE-E extract. There was no difference in the antioxidant properties among different extracts. In vitro human liver microsome assays illustrated that all extracts possessed herb-drug interaction potentials but the UE-E extract are shown with a potent interaction with CYP3A4-metabolized drugs. CONCLUSION: MAE-W is a favorable method for the preparation of HQ extracts based on extraction yield, pharmacological properties and safety.