Nitin Kishor (Presenter)
INDIAN INSTITUTE OF SCIENCE EDUCATION AND RESEARCH
Bio: Personal bio sketch – My name is Nitin Kishor. My birth place is Bhagalpur (Bihar, India) and date of birth is 30th of July 1983. I did BSC in Zoology. After that I did MSC in Biochemistry. Currently I am in Ph.D final year from Indian Institute of Science Education and Research Mohali, India. My specialization area is Protein Biochemistry in which I study protein folding, stability, aggregation, structure and function. I have intense desire in mass spectrometry. I am using mass spectrometry as a tool for identification through intact mass and peptide mass fingerprinting and analysis and searching of protein through database by using raw mass spectrometry data. Currently, I am life member of Indian society of mass spectrometry (ISMAS) and Proteomics Society of India (PSI). Through the conference, I will have golden chance of exposure of mass spectrometry in clinical labs.
Authorship: A. Nitin Kishor and B. Purnananda Guptasarma
Department of Biological Science, Indian Institute of Science Education and Research Mohali (IISER MOHALI)
| Short Abstract Aminopeptidases catalyze the hydrolysis of peptide bonds joining the N-terminal amino acid of any peptide to the next amino acid in the sequence. Processive aminopeptidase can be used to create a population of peptides of different lengths differing by one amino acid mass, so that the entire population can be studied in a single mass spectrum, to determine the peptide’s amino acid sequence. Sometimes, structure present in the peptide can also interfere. In this study, we describe the use of a non-specific, processive, deblocking Bacillus subtillis-derived aminopeptidase (BsuAP) in a reaction. |
Long Abstract
1. Introduction
Aminopeptidases are exo-peptidases that catalyze the hydrolysis of peptide bonds joining the N-terminal amino acid of any peptide to the next amino acid in the sequence. Non- processive aminopeptidases are specific for certain residues and cannot progressively hydrolyze successively placed amino acid residues, whereas processive aminopeptidases tend to be non-specific and can ‘progressively’ cleave up to 30-35 amino acid residues in any peptide [1,2]. Theoretically, therefore, beginning with a homogenous population of molecules of a defined mass constituting a single peptide of defined amino acid sequence, any processive aminopeptidase can be used to create a population of peptides of different lengths differing by one amino acid mass, so that the entire population can be studied in a single mass spectrum, to determine the peptide’s amino acid sequence. However, in practice, it is difficult to control the reaction from progressing rapidly and also some peptides have blocked N-termini which prevent facile proteolysis. Sometimes, structure present in the peptide can also interfere. In this study, we describe the use of a non-specific, processive, deblocking Bacillus subtillis-derived aminopeptidase (BsuAP) in a reaction conducted at 70 degrees Centigrade for N-terminus sequencing.
2. Method
Glu fibrino peptide (GFP B) from Waters, USA, was used as substrate. GFP B is used as a standard (lock mass) in mass spectrometry and has a mass of 1570.67 Da. BsuAP was a recombinant protein expressed in, and purified from, E. coli in our own lab [3]. The reaction mixture of enzyme and substrate was prepared by mixing 1 nanomole of the aminopeptidase to 1.6 nanomoles of the substrate (GFP B) in water. The reaction mixture and control reaction (lacking aminopeptidase) were incubated at 700c for 1 hour. After incubation, the reaction-mixture and control reaction were spotted on the MALDI plate. GFP B was used as a lock mass control. For spotting, 1µl of sample was spotted with 1µl CHCA matrix. Samples were analyzed on the Q-TOF Synapt G2S HDMS system (from Waters, USA), with laser desorption achieved by the instrument’s 355nm laser. The spectra analyzed and presented are lock mass-corrected spectra.
3. Result and Conclusion
The GFP B peptide is 14 residues-long. A sufficiently slow reaction would produce a mass spectrum containing the mass corresponding to the complete peptide, and all smaller masses progressively truncating the peptide from its N-terminus, assuming that the reaction does not proceed to completion on all substrate molecules. Our data suggests that under the conditions used, BsuAP is non-specific and quite processive, progressively digesting 11 amino acid residues in the peptide substrate, beginning with the N-terminal glycine. Masses are seen to have different relative intensities which could be indicative of differences in specificity. It may be noted that the differences in masses of successive peaks (moving leftwards from the control GFP B mass) correspond to masses of the amino acids removed, after addition of the mass of one hydrogen.
References & Acknowledgements:
References:-
1. B.Franzetti, G.Schoehn ,J.-F.Hernandez, M.Jaquinod , R.W.H.Ruigrok and G.Zaccai, The EMBO Journal. Vol. 21 :9 ( 2002) 2132-2138.
2. M. Asuncio Dura, Veronique Receveur-Brechot, Jean-Pierre Andrieu, Christine Ebel, Guy Schoehn, Alain Roussel, and Brun,. Biochemistry 44 (2005) 3477-3486.
3. D.Kapoor, B.Singh, S.Karthikeyan, and P.Guptasarma, Enzyme and Microbial Technology 46 (2010) 1–8.
4. N. Kishor and P.Guptasarma, Analytical Biochemistry 488 (2015) 6-8.
Acknowledgements:-
IISER Mohali for research funding in PG’s lab. UGC for research fellowship support to NK.
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