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Understanding the Proteomics of Medicinal Plants under Environmental Pollution
Published in Azamal Husen, Environmental Pollution and Medicinal Plants, 2022
Pooja Singh, V.K. Mishra, Rohit Kashyap, Rahul Rawat
The protein spots obtained in 2-DE are excised from 2-D gel and then the target protein is digested with appropriate protease into peptide fragments. Then a set of masses of these peptides is constructed by MS such as MALDI-TOF or ESI-TOF, called ‘peptide mass fingerprinting’ (PMF) which is characteristic for the target protein and is used to search peptide masses generated by theoretical fragmentation of protein sequences of databases (Yates 1998b). Matches from at least three to six peptides derived from the same protein are required to positively identify a protein (Pappin et al. 1993; Yates 1998b).
Genetics of chronic pain: crucial concepts in genetics and research tools to understand the molecular biology of pain and analgesia
Published in Peter R Wilson, Paul J Watson, Jennifer A Haythornthwaite, Troels S Jensen, Clinical Pain Management, 2008
Bradley E Aouizerat, Christine A Miaskowski
More recently, mass spectroscopy promises to advance the field of proteomics. Two different methods of mass spectroscopy are being applied: peptide mass fingerprinting and tandem mass spectroscopy. Peptide mass fingerprinting identifies a protein by cleaving it into short peptides and uses a peptide sequence database to align the short peptides and then deduce the protein’s identity by matching it against proteins in the database. By comparison, tandem mass spectrometry derives sequence information from individual peptides that are isolated and then collided with a nonreactive gas. Data on the array of fragment ions produced is recorded and analyzed. Unlike genome- and transcriptome-based methods, proteomic analyses struggle to attain the same level of throughput. The major limitation is that proteins cannot be amplified in a manner similar to nucleic acid amplification and the cost of mass spectroscopy is prohibitive.
Applications of MALDI-TOF mass spectrometry in clinical proteomics
Published in Expert Review of Proteomics, 2018
Viviana Greco, Cristian Piras, Luisa Pieroni, Maurizio Ronci, Lorenza Putignani, Paola Roncada, Andrea Urbani
Proteins identification is carried out by the so-called peptide mass mapping based on the accurate mass measurement of a mixture of peptides derived from a protein. Through a bottom-up approach, peptide masses are detected by MALDI-TOF MS. The spectrum is unique for that specific protein (Peptide Mass Fingerprinting, PMF). Identification is complemented by the search for peptide mass values against a database of proteolytic peptide masses calculated by known protein sequences (e.g. NCBI, SwissProt, TrEMBL). This approach ensures robust identification; however, the confidence of the search and consequently the reliability of identification decrease if more proteins are present in the same sample, requiring further MS/MS-based analysis.
Bone morphogenetic protein (BMP) 7 expression is regulated by the E3 ligase UBE4A in diabetic nephropathy
Published in Archives of Physiology and Biochemistry, 2020
Ying Feng, Ming-Yue Jin, Dong-Wei Liu, Li Wei
Mass spectrometry: Silver stained bands were excised and submitted to Beijing MDL Biotechnology Company for further processing using standard protocols. Matrix-assisted laser desorption/ionization time-of flight (MALDI-TOF) mass spectrometer (Voyager-DE PRO, ThermoFisher Scientific) was used for peptide mass fingerprinting at a local core facility. Peptides were analyzed using the MASCOT algorithm. Prioritization of putative E3 ligases to be subsequently tested was done based on peptide coverage threshold of 40%.
Differential levels of CHMP2B, LLPH, and SLC25A51 proteins in secondary renal amyloidosis
Published in Expert Review of Proteomics, 2021
Nimisha Gupta, Tahreem Sahar, Sapna Khowal, Ishfaq Ahmad Ganaie, Mohd Mughees, Dinesh Khullar, S. K. Jain, Saima Wajid
The differential spots of interest were excised from the 2DE gels by spot picker and placed into Eppendorf in milliQ. The gel pieces were decolorized with the destaining solution (50 mM sodium thiosulfate and 15 mM potassium ferricyanide) and subsequent washes with milliQ. Gel pieces were dehydrated with 30% acetonitrile (ACN) and air dried with Speedvac. Gel pieces were rehydrated according to the previously published study [12]. The gel pieces were soaked with Iodoacetamide (100 mM) for 45 min. The supernatant was removed and the gel was allowed to incubate in ammonium bicarbonate solution for 10 minutes. The trypsin solution was added and digested for 16 h at 37 °C. The digested solution was passed to fresh microcentrifuge tubes. The gel pieces were extracted thrice with extraction buffer and the supernatant was collected into the microcentrifuge tube and then dried with Speedvac. The dried peptide mixture was suspended in Tris-acetate buffer (20 mM, pH 7.5) and mixed with HCCA matrix followed by spotting onto the MALDI ground steel target plate. MALDI plate was dried by air and further analysis was done by MALDI-TOF Ultraflex III instrument (Bruker Daltonics). The fragmented peptide was analyzed with FLEX ANALYSIS SOFTWARE (Version 3.3) to obtain the PEPTIDE MASS FINGERPRINT (PMF). The Peptide mass tolerance for precursor ions varied for each spot as ± 80 ppm (SSP 5108), ± 100 ppm (SSP 6109), ± 160 ppm (SSP 8108), ± 1.25 Da (SSP 1103), ± 1.5 Da (SSP 2102), ± 1.5 Da (SSP 1108), ± 1.5 Da (SSP 3003), ± 1.45 Da (SSP 8209), ± 1.45 Da (SSP 9402), ± 1.45 Da (SSP 7106), and ± 1.25 Da (SSP 7307). Peptide masses and fragment masses were compared with the theoretical masses of all proteins using human taxonomy in the SWISS-PROT and National Centre of Biotechnology nonredundant (NCBInr) databases. Then, the masses were submitted to MASCOT search engine for Peptide Mass Fingerprinting analysis to characterize the protein.