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• Development of a simple single step method for extraction of proteins from mammalian cell lines and tissue samples. • Developed method is compatible with gel based and gel free approaches. • Developed protocol is useful for a wide range of studies involving various biological samples. • Developed method is rapid and provides high yield and better proteome coverage. • Mass spectrometry compatible protein extraction method. Proteins are crucial research molecules in modern biology. Almost every biological research area needs protein-based assays to answer the research questions. The study of the total protein content of a biological sample known as Proteomics, is one of the highly rated qualitative and quantitative approach to address numerous biological problems including clinical research. The key step to successfully generate high quality proteomics data is the efficient extraction of proteins from biological samples. Although different methods are in use for protein extraction from a wide variety of samples, however, because of their prolonged protocol and multiple steps involved, final protein yield is sacrificed. Here, we have shown the development of a simple single step method for extraction of proteins from mammalian cell lines as well as tissue samples in an effective and reproducible manner. This method is based on lysis of samples directly in a modified lysis buffer without CHAPS (7 M Urea, 2 M Thiourea, and 10 mM Tris-Cl; pH 8.5) that is compatible with gel based and gel free approaches. This developed protocol is reliable and should be useful for a wide range of proteomic studies involving various biological samples. [ABSTRACT FROM AUTHOR]

Infections caused by Mycobacterium tuberculosis and nontuberculous mycobacteria represent a significant global threat and medical concern. Therefore, accurate and reliable methods must be employed to identify mycobacteria rapidly. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a technique that compares the cellular protein profiles of unknown isolates with reference mass spectra in a database to identify microorganisms. However, the thick and waxy lipid layer, which is rich in mycolic acids and is present in mycobacterial cells, makes protein extraction challenging. To identify the optimal protocol for correctly identifying bacilli using MALDI-TOF mass spectrometry, this study compared four different cellular protein extraction methods. Four strains of M. bovis BCG were selected as representatives of slow-growing mycobacteria, while three strains of fast-growing mycobacteria were also included: M. peregrinum , M. smegmatis, and M. farcinogenes. The extraction method that proved most effective was the extraction of inactivated cells with chloroform and methanol, which partially delipidates the cells. These cells were then extracted with formic acid, as is standard practice for protein extraction. The advantage of this method is that it allows the parallel analysis of cellular lipids and proteins from a single sample. It is therefore important to optimize mycobacterial protein extraction for MALDI-TOF MS analysis in clinical microbiology laboratories. • This study aimed to find the best protein extraction protocol for identifying mycobacteria using MALDI-TOF MS. • The most effective protocol involves additional extraction of cells using chloroform and methanol to remove lipids partially. • It allows for simultaneous analysis of cellular proteins and lipids using the same sample • The choice of the medium had minimal impact on MALDI-TOF Biotyper identification results • Proper optimisation of mycobacterial protein extraction is critical when using MALDI-TOF MS. [ABSTRACT FROM AUTHOR]

Banana is one of the most important crops, providing multiple benefits. Although it has been widely studied for its health benefits, little information can be found about its proteins. This study determined the antihypertensive and antioxidant activities of the crude, purified, and hydrolyzed protein extracts from 'Señorita' banana pulp. Crude proteins were extracted using Tris-HCl buffer and purified through ammonium sulfate precipitation, dialysis, and gel filtration chromatography. The protein content of the crude, partially purified, and purified extracts were found to be 167.32, 120.45, and 28.51 μg·mL−1, respectively, with major protein having an approximate molecular weight of 15 kDa. These extracts were then subjected to enzymatic hydrolysis for release of bioactive peptides prior to ACE inhibitory and antioxidant activities determination. Among these samples, the undigested crude extract had the highest ACE inhibitory activity (85.20%). There was also an observable increase in ACE inhibition of the digested samples with increased digestion time. Meanwhile, the 3-h and 4-h crude digests had the highest DPPH radical scavenging activity with 30.82% and 34.74%, respectively. These were not significantly different from the activity of the standard, ascorbic acid. A general decrease in DPPH radical scavenging activity of the samples was observed with increased digestion time. These observations were coherent with the in silico analysis of the putative major protein, lectin, which showed that its enzymatic hydrolysis releases ACE inhibitor and antioxidant peptides. [ABSTRACT FROM AUTHOR]

Banana is one of the most important crops, providing multiple benefits. Although it has been widely studied for its health benefits, little information can be found about its proteins. This study determined the antihypertensive and antioxidant activities of the crude, purified, and hydrolyzed protein extracts from ‘Señorita’ banana pulp. Crude proteins were extracted using Tris-HCl buffer and purified through ammonium sulfate precipitation, dialysis, and gel filtration chromatography. The protein content of the crude, partially purified, and purified extracts were found to be 167.32, 120.45, and 28.51 μg·mL−1, respectively, with major protein having an approximate molecular weight of 15 kDa. These extracts were then subjected to enzymatic hydrolysis for release of bioactive peptides prior to ACE inhibitory and antioxidant activities determination. Among these samples, the undigested crude extract had the highest ACE inhibitory activity (85.20%). There was also an observable increase in ACE inhibition of the digested samples with increased digestion time. Meanwhile, the 3-h and 4-h crude digests had the highest DPPH radical scavenging activity with 30.82% and 34.74%, respectively. These were not significantly different from the activity of the standard, ascorbic acid. A general decrease in DPPH radical scavenging activity of the samples was observed with increased digestion time. These observations were coherent with the in silico analysis of the putative major protein, lectin, which showed that its enzymatic hydrolysis releases ACE inhibitor and antioxidant peptides.

Plant viruses are important pathogens that cause significant crop losses. A plant protein extraction protocol that combines crushing the tissue by a pestle in liquid nitrogen with subsequent crushing by a roller-ball crusher in urea solution, followed by RuBisCO depletion, reduction, alkylation, protein digestion, and ZipTip purification allowed us to substantially simplify the sample preparation by removing any other precipitation steps and to detect viral proteins from samples, even with less than 0.2 g of leaf tissue, by a medium resolution nanoLC-ESI-Q-TOF. The presence of capsid proteins or polyproteins of fourteen important viruses from seven different families (Geminiviridae, Luteoviridae, Bromoviridae, Caulimoviridae, Virgaviridae, Potyviridae, and Secoviridae) isolated from ten different economically important plant hosts was confirmed through many identified pathogen-specific peptides from a protein database of host proteins and potential pathogen proteins assembled separately for each host and based on existing online plant virus pathogen databases. The presented extraction protocol, combined with a medium resolution LC-MS/MS, represents a cost-efficient virus protein confirmation method that proved to be effective at identifying virus strains (as demonstrated for PPV, WDV) and distinct disease species of BYDV, as well as putative new viral protein sequences from single-plant-leaf tissue samples. Data are available via ProteomeXchange with identifier PXD022456.

Abstract Mass spectrometry is the most used method for protein identification and quantification. Here we developed four protein extraction protocols precisely for mass spectrometry, and we compared with other ones already published. The best protocol developed by us consists on a simple extraction solution, a heat-shock step, and does not use protease inhibitor; moreover, it is the most efficient and uniform among replicates, besides to be safe, cheap and fast. That method also provided the highest number of proteins uniquely identified and allows finding a diversity of protein classes, which their absence is a problem to be avoided. Highlights • Liquid nitrogen and mechanic strength provided the easiest way to reach intracellular proteins. • The protocol 6 gave the highest amount of proteins, but it is not the most replicate-consistent. • The protocol 7 is very consistent, easy, provides most of unique proteins and a variety of classes. [ABSTRACT FROM AUTHOR]

Abstract: A proteomic analysis was conducted on peach fruit mesocarp in order to better elucidate the biochemical and physiological events which characterize the transition of fruit from the “unripe” to the “ripe” phase. The first goal of the present work was to set-up a protocol suitable for improving protein extraction from peach mesocarp. The use of freeze-dried powdered tissue, together with the addition of phenol prior to the extraction with an aqueous buffer, significantly increased the protein yield and the quality of 2-DE gels. The proteomic profiles of the mesocarp from peach fruit of a non-melting flesh (NMF; ‘Oro A’) and a melting flesh (MF; ‘Bolero’) cultivar, at “unripe” and “ripe” stages as defined by some parameters typical of ripening, were then analyzed. The comparative analysis of the 2-DE gels showed that in NMF and MF peaches the relative volumes of 53 protein spots significantly changed in relation to both the ripening stage (“unripe” versus “ripe”) and/or the genetic background of the cultivar (‘Oro A’ versus ‘Bolero’). Thirty out of the 53 differently abundant spots were identified by LC–ESI-MS/MS. The analysis revealed enzymes involved in primary metabolism (e.g. C-compounds, carbohydrates, organic acids and amino acids) and in ethylene biosynthesis as well as proteins involved in secondary metabolism and responses to stress. Among these, 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) appeared to be one of the proteins with the largest change in relative abundance during the fruit transition from the pre-climacteric (“unripe”) to the climacteric (“ripe”) phase. Other proteins, such as S-adenosylmethionine synthetase and β-cyanoalanine synthase involved in ethylene metabolism, were also identified. Moreover, the changes in the relative abundances of a sucrose synthase and an α-amylase suggested differences between the two cultivars in the carbohydrate import activity of ripe fruit. The different accumulation of a few typical ROS-scavenger enzymes suggested that a higher oxidative stress occurred in MF with respect to NMF fruit. This result, together with data concerning the levels of total proteins and free amino acids and those regarding proteins involved in the maintenance of tissue integrity, was consistent with the hypothesis that the last phase of ripening in MF fruit is characterized by the appearance of a senescence status. The present study appears to define well some of the biochemical and physiological events that characterize the ripening of peach and, at the same time, provides interesting indications that could be employed in future marker assisted selection (MAS) programmes aimed to obtain MF fruits with higher ability to preserve tissue functionality maintaining for a longer time their organoleptic characteristics. [Copyright &y& Elsevier]

Purpose: Trichosporon species are emerging human pathogens, accounting for the second most common cause of non-candidal mycosis. Rapid and reliable identification of these agents allows a better understanding of their epidemiology and therapeutic management. The Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) technique has the potential to be precise, fast and cost-effective. However, the precision of identification totally depends upon the type of protein extraction method used and embedded database in the system. Our objectives were to standardize the protein extraction technique and expand the present Bruker database by creating an in-house database and validating it with diverse clinical Trichosporon species of Indian origin., Methods: Two different protein extraction protocols (on-plate and off-plate) were evaluated. The off-plate protocol was finalized for the identification. MALDI TOF MS with the existing Bruker database was evaluated for its ability to identify a total of 79 intergenic spacer 1 (IGS1) gene sequence confirmed clinical isolates of 5 different Trichosporon species., Results: As outcome, off plate protocol yielded higher accuracy (73% on the species level and 95% on the genus level) than on-plate (25% on the genus level) in terms of log scores. The existing database for Trichosporon species was enriched with 28 sequence confirmed isolates, which improved accuracy from 73% to 100% and were identified up to species level with a log score >2.3., Conclusions: Used with standardized protein-extraction protocol along with an expanded database, MALDI-TOF MS could be a rapid and reliable approach to identify clinical Trichosporon species routinely in the laboratory., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)

Plant viruses are important pathogens that cause significant crop losses. A plant protein extraction protocol that combines crushing the tissue by a pestle in liquid nitrogen with subsequent crushing by a roller-ball crusher in urea solution, followed by RuBisCO depletion, reduction, alkylation, protein digestion, and ZipTip purification allowed us to substantially simplify the sample preparation by removing any other precipitation steps and to detect viral proteins from samples, even with less than 0.2 g of leaf tissue, by a medium resolution nanoLC-ESI-Q-TOF. The presence of capsid proteins or polyproteins of fourteen important viruses from seven different families (Geminiviridae, Luteoviridae, Bromoviridae, Caulimoviridae, Virgaviridae, Potyviridae, and Secoviridae) isolated from ten different economically important plant hosts was confirmed through many identified pathogen-specific peptides from a protein database of host proteins and potential pathogen proteins assembled separately for each host and based on existing online plant virus pathogen databases. The presented extraction protocol, combined with a medium resolution LC-MS/MS, represents a cost-efficient virus protein confirmation method that proved to be effective at identifying virus strains (as demonstrated for PPV, WDV) and distinct disease species of BYDV, as well as putative new viral protein sequences from single-plant-leaf tissue samples. Data are available via ProteomeXchange with identifier PXD022456. [ABSTRACT FROM AUTHOR]

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has proved to be a useful diagnostic method for identifying conventional bacteria. In the case of mycobacteria, a good protein extraction protocol is essential in order to obtain reliable identification results. To date, no such protocol has been definitively established. The aim of this study was to compare the manufacturer's recommended protein extraction protocol (protocol A) with two novel protocols (protocols B and C), which apply different freezing temperatures and mechanical disruption times using an automatic tissue homogenizer. A total of 302 clinical isolates, comprising 41 nontuberculous mycobacteria (NTM) species, were grown in parallel on solid and liquid media and analyzed: 174 isolates were slow-growing mycobacteria (SGM) and 128 isolates were rapid-growing mycobacteria (RGM). Overall, MALDI-TOF MS identified a higher number of NTM isolates from solid than from liquid media, especially with protocol C (83.4 and 68.2%, respectively; P < 0.05). From solid media, this protein extraction method identified 57.9 and 3.9% more isolates than protocols A ( P < 0.001) and B ( P < 0.05), respectively. In the case of liquid media, protocol C identified 49.7 and 6.3% more isolates than protocols A and B, respectively ( P < 0.001). With regard to the growth rate, MALDI-TOF MS identified more RGM isolates than SGM isolates in all of the protocols studied. In conclusion, the application of freezing and automatic tissue homogenizer improved protein extraction of NTM and boosted identification rates. Consequently, MALDI-TOF MS, which is a cheap and simple method, could be a helpful tool for identifying NTM species in clinical laboratories., (Copyright © 2018 American Society for Microbiology.)