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MS‐based proteomics is expanding its role as a routine tool for biological discovery. Nevertheless, the task of accurately and precisely quantifying thousands of analytes in a single experiment remains challenging. In this study, the diagnostic accuracy of three popular data‐dependent methods for protein relative quantification (label‐free [LF], dimethyl labelling [DML] and tandem mass tags [TMT]) has been assessed using a mixed species proteome (three species) and five experimental replicates per condition. Data were produced using a quadrupole‐Orbitrap mass spectrometer and analysed using a single platform (the MaxQuant/Perseus software suite). The whole comparative analysis was repeated three times over a period of 6 months, in order to assess the consistency of the reported findings. As expected, label‐based methods reproducibly provided a lower false positives rate, whereas TMT and LF performed similarly, and significantly better than DML, in terms of proteome coverage using the same instrument time. Although parameters like proteome coverage and precision were consistent in between replicates, other parameters like sensitivity, intended as the capacity of correctly classifying true positives (regulated proteins), were found to be less reproducible, especially at challenging fold‐changes (1.5). Collectively, data suggest that an increased interest in data reproducibility would be desirable in the quantitative proteomics field. [ABSTRACT FROM AUTHOR]

Herein we present a simple, reproducible and versatile approach for in situ protein digestion and identification on formalin-fixed and paraffin-embedded ( FFPE) tissues. This adaptation is based on the use of an enzyme delivery platform (hydrogel discs) that can be positioned on the surface of a tissue section. By simultaneous deposition of multiple hydrogels over select regions of interest within the same tissue section, multiple peptide extracts can be obtained from discrete histological areas. After enzymatic digestion, the hydrogel extracts are submitted for LC- MS/ MS analysis followed by database inquiry for protein identification. Further, imaging mass spectrometry ( IMS) is used to reveal the spatial distribution of the identified peptides within a serial tissue section. Optimization was achieved using cutaneous tissue from surgically excised pressure ulcers that were subdivided into two prime regions of interest: the wound bed and the adjacent dermal area. The robust display of tryptic peptides within these spectral analyses of histologically defined tissue regions suggests that LC- MS/ MS in combination with IMS can serve as useful exploratory tools. [ABSTRACT FROM AUTHOR]

This study presents on-tissue proteolytic digestion using a microwave irradiation and peptide extraction method for in situ analysis of proteins from spatially defined regions of a tissue section. The methodology utilizes hydrogel discs (1 mm diameter) embedded with trypsin solution. The enzyme-laced hydrogel discs are applied to a tissue section, directing enzymatic digestion to a spatially confined area of the tissue. By applying microwave radiation, protein digestion is performed in 2 min on-tissue, and the extracted peptides are then analyzed by matrix assisted laser desorption/ionization mass spectrometry (MALDI MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS). The reliability and reproducibility of the microwave assisted hydrogel mediated on-tissue digestion is demonstrated by the comparison with other on-tissue digestion strategies, including comparisons with conventional heating and in-solution digestion. LC-MS/MS data were evaluated considering the number of identified proteins as well as the number of protein groups and distinct peptides. The results of this study demonstrate that rapid and reliable protein digestion can be performed on a single thin tissue section while preserving the relationship between the molecular information obtained and the tissue architecture, and the resulting peptides can be extracted in sufficient abundance to permit analysis using LC-MS/MS. This approach will be most useful for samples that have limited availability but are needed for multiple analyses, especially for the correlation of proteomics data with histology and immunohistochemistry. [ABSTRACT FROM AUTHOR]

ABSTRACT Introduction Vegetables contain a variety of phytochemicals that have the ability to modify enzymatic and chemical reactions, and therefore may have a positive influence on human health. In particular kaempferol is known to possess anti-carcinogenic activity. Objective The purpose of this work was to determine the structure of glycosylated kaempferol derivatives, acylated with malonic acid on the sugar portion. Methods A methanolic extract of the leaves of Allium porrum L. was submitted to fractionation procedures through semi-preparative HPLC/UV-MS techniques. The collected fractions were evaluated by accurate tandem mass spectrometry experiments using an electrospray ionisation (ESI) quadrupole time-of-flight instrument. Isolated compounds were hydrolysed in order to obtain information on the ester moieties. Results The structures of five compounds not previously reported in leek were determined. The molecules are mono-hexose, di-hexose and coumaroyl, feruloyl and caffeoyl acylated di-hexose derivatives of kaempferol. The common characteristic of the structures relies on the presence of the malonyl moiety on the primary alcoholic function of the sugar immediately linked to the aglycone. Accurate tandem MS experiments and basic hydrolysis treatments revealed a sequence of the acylated glycosidic moieties. Conclusion A set of secondary metabolites of the aerial part of Allium porrum L. (leek) was identified and characterised by ESI/MS2. Knowledge of the presence of these first-reported compounds in leek could provide the means for fully understanding of the metabolism of this plant in relation to the biosynthesis of the phenolics. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Purpose: The application of a methodology for quantitative protein analysis from formalin-fixed and paraffin-embedded (FFPE) tissue by using hydrogels. Miniaturized polymeric gels are placed onto histologically defined tissue regions in order to perform localized digestion for bottom-up proteomics. Hydrogel-extracted peptides are then labeled with tandem mass tags (TMT) reagents for relative protein quantification. A cardiac myxoma biopsy is used., Experimental Design: Multiple hydrogels, incorporating the proteolytic enzyme trypsin, are placed on serial tissue sections, and processed for digestion and TMT derivatization. SCX fractionation before LC-MS/MS analysis and bioinformatics analysis are carried out., Results: Two histologically different areas on both FFPE and frozen sections of the same cardiac myxoma biopsy are compared. In total, 1949 (FFPE) and 2491 (frozen) proteins are identified, with a total overlap of 56%. The quantitative comparison highlighted 15 (FFPE) and 138 (frozen) differentially expressed proteins between myxoma regions., Conclusion: The methodology successfully detects numerous protein signals from FFPE and frozen specimens and is able to differentiate between tissue regions. A fast and reliable tissue preparation for quantitative protein analysis by minimum sample manipulation is developed. This offers an option for on-tissue proteomics analysis while preserving the inherent spatial information on the tissue., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)