01963nas a2200229 4500008004100000022001400041245014700055210006900202260007300271300001200344490000800356520107500364653001201439653002501451653002201476100001901498700002001517700001901537700002501556700002201581856013001603 2007 eng d a0168-165600aCharacterization of the proteins of bacterial strain isolated from contaminated site involved in heavy metal resistance - a proteomic approach0 aCharacterization of the proteins of bacterial strain isolated fr aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcFEB a444-4510 v1283 a

The present study describes response of a bacterial strain isolated from a polluted river to heavy metal toxicity. The bacterium was identified to be Klebsiella pneumoniae by biochemical tests using API 20E strips and 16S ribotyping. The isolate was studied for its tolerance to two heavy metals, i.e., cobalt (Co2+) and lead (Pb2+) by growing it in citrate mineral medium (CMM). Proteomic approach involving two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and mass spectrometry (MS) was used to identify the differentially expressed proteins under heavy metal stress. Two of the differentially expressed proteins were identified to be L-isoaspartate protein carboxymethyltransferase type II and DNA gyrase A. To our knowledge, this is for the first time that K pneumoniae has been reported to be present in metal contaminated site and L-isoaspartate protein carboxymethyltransferase type II protein to be over expressed under heavy metal stress. The role of these proteins in metal tolerance is discussed. (c) 2006 Elsevier B.V. All rights reserved.

10a2D PAGE10aheavy metal toxicity10aMass spectrometry1 aBar, Chinmayee1 aPatil, Rajendra1 aDoshi, Jignesh1 aKulkarni, Mahesh, J.1 aGade, Wasudeo, N. uhttps://library.ncl.res.in/content/characterization-proteins-bacterial-strain-isolated-contaminated-site-involved-heavy-metal02720nas a2200289 4500008004100000022001400041245015100055210006900206260008100275300002200356490000700378520163500385653001302020653001402033653001402047653002102061653001002082653002202092100003902114700002502153700002502178700002002203700002302223700002902246700002502275856013002300 2016 eng d a1542-641600aTargeted quantification of N-1-(carboxymethyl) valine and N-1-(carboxyethyl) valine peptides of beta-hemoglobin for better diagnostics in diabetes0 aTargeted quantification of N1carboxymethyl valine and N1carboxye a236 GRAYS INN RD, FLOOR 6, LONDON WC1X 8HL, ENGLANDbBIOMED CENTRAL LTDcMAR aArticle Number: 70 v133 a

Background: N-1-(Deoxyfructosyl) valine (DFV) beta-hemoglobin (beta-Hb), commonly referred as HbA1c, is widely used diagnostic marker in diabetes, believed to provide glycemic status of preceding 90-120 days. However, the turnover of hemoglobin is about 120 days, the DFV-beta-Hb, an early and reversible glycation product eventually may undergo irreversible advanced glycation modifications such as carboxymethylation or carboxyethylation. Hence quantification of N-1-(carboxymethyl) valine (CMV) and N-1-(carboxyethyl) valine (CEV) peptides of beta-Hb would be useful in assessing actual glycemic status. Results: Fragment ion library for synthetically glycated peptides of hemoglobin was generated by using high resolution-accurate mass spectrometry (HR/AM). Using parallel reaction monitoring, deoxyfructosylated, carboxymethylated and carboxyethylated peptides of hemoglobin were quantified in clinical samples from healthy control, pre-diabetes, diabetes and poorly controlled diabetes. For the first time, we report N-1-beta-valine undergoes carboxyethylation and mass spectrometric quantification of CMV and CEV peptides of beta-hemoglobin. Carboxymethylation was found to be the most abundant modification of N-1-beta-valine. Both CMV-beta-Hb and CEV-beta-Hb peptides showed better correlation with severity of diabetes in terms of fasting glucose, postprandial glucose and microalbuminuria. Conclusions: This study reports carboxymethylation as a predominant modification of N-1-beta-valine of Hb, and quantification of CMV-beta-Hb and CEV-beta-Hb could be useful parameter for assessing the severity of diabetes.

10aDiabetes10aDiagnosis10aGlycation10aGlycemic control10aHbA1c10aMass spectrometry1 aJagadeeshaprasad, Mashanipalya, G.1 aBatkulwar, Kedar, B.1 aMeshram, Nishita, N.1 aTiwari, Shalbha1 aKorwar, Arvind, M.1 aUnnikrishnan, Ambika, G.1 aKulkarni, Mahesh, J. uhttps://library.ncl.res.in/content/targeted-quantification-n-1-carboxymethyl-valine-and-n-1-carboxyethyl-valine-peptides-beta02333nas a2200289 4500008004100000022001400041245007800055210006900133260000800202300001100210490000800221520142300229653001401652653002401666653002401690653002201714653003301736653002201769100001801791700001601809700002401825700002201849700002001871700002501891700002001916856010701936 2024 eng d a0141-813000aMulti-dimensional impact of captopril modification on human serum albumin0 aMultidimensional impact of captopril modification on human serum cAUG a1332890 v2743 a

Captopril is a thiol drug, widely used for the management of hypertension and cardiovascular diseases. Reactive thiols are found to covalently modify the cysteines of plasma proteins and affect their structure and function. Human serum albumin (HSA) is prone to undergo modification by various low molecular weight compounds, including drugs. Cysteine34 (Cys34) in HSA has a free thiol group with antioxidant properties, considered to be the most redox-sensitive amino acid in plasma. Through mass-spectrometric analysis, we demonstrate for the first time that captopril forms a disulfide adduct at Cys34 residue and increases the protease susceptibility of HSA to trypsin. As evidenced by our biophysical and electron microscopy studies, HSA undergoes structural alteration, aggregation and morphological changes when treated with different captopril concentrations. Molecular dynamics studies further revealed the regions of secondary structural changes in HSA due to disulfide adduct formation by captopril at Cys34. It also elucidated the residues involved in the noncovalent interactions with captopril. It is envisaged that structural change in HSA may influence the efficacy of drug delivery as well as its own biological function. These findings may thus provide significant insights into the field of pharmacology intriguing further investigation into the effects of long-term captopril treatment.

10aCaptopril10aElectron microscopy10aHuman serum albumin10aMass spectrometry10aMolecular dynamic simulation10aSpectrophotometry1 aGhosh, Amrita1 aJani, Vinod1 aSonavane, Uddhavesh1 aNaphade, Anvi, N.1 aJoshi, Rajendra1 aKulkarni, Mahesh, J.1 aGiri, Ashok, P. uhttps://library.ncl.res.in/content/multi-dimensional-impact-captopril-modification-human-serum-albumin02205nas a2200241 4500008004100000022001400041245009500055210006900150260000800219300001200227490000700239520141000246653002001656653002301676653001201699653002201711100002301733700002301756700002301779700001501802700002501817856012101842 2025 eng d a1572-388700aCharacterization of S-homocysteinylation of Human Insulin and Its Implications in Diabetes0 aCharacterization of Shomocysteinylation of Human Insulin and Its cOCT a598-6100 v443 a

Homocysteine thiolactone is a reactive thiol known for its interaction with various proteins. Nevertheless, there exists a paucity of information concerning the interaction between homocysteine thiolactone and human insulin, particularly regarding the mechanism by which homocysteine facilitates the reduction of disulfide bonds within insulin. In the present study, we have elucidated the binding sites of homocysteine to the cysteine residues (A6-B7 and A20-B19) that are implicated in the formation of intermolecular disulfide bonds in insulin through an in vitro reaction analyzed via LC-ESI MS/MS. This results in a reduction of disulfide bonds linking the A and B chains, which was corroborated by MALDI-TOF-MS and ESI-MS analysis. The secondary structure of insulin is affected by this modification, as evidenced by circular dichroism spectroscopy. In-silico studies also show that homocysteine affects the insulin structure. A glucose uptake assay conducted in Chinese hamster ovary (CHO) cells that stably express the insulin receptor revealed that HC-modified insulin is less effective in inducing glucose uptake compared to native insulin, suggesting that HC-induced structural modifications in insulin influence functional activity. This study provides insight into the HC-induced structural and functional changes in insulin and discusses the consequent implications for diabetes.

10aDisulfide bonds10aHomocysteinylation10ainsulin10aMass spectrometry1 aSheikh, Amreen, B.1 aJathar, Swaraj, M.1 aTammara, Vaishnavi1 aDas, Atanu1 aKulkarni, Mahesh, J. uhttps://library.ncl.res.in/content/characterization-s-homocysteinylation-human-insulin-and-its-implications-diabetes02515nas a2200277 4500008004100000022001400041245011700055210006900172260000800241300001100249490000800260520162800268653001301896653002201909653001201931653001201943653002201955100001501977700001901992700002002011700001902031700002002050700001902070700002502089856012302114 2025 eng d a1046-592800aSimple freeze-thaw based method for efficient purification of recombinant human proinsulin from inclusion bodies0 aSimple freezethaw based method for efficient purification of rec cMAR a1066450 v2273 a

Insulin is a pivotal peptide hormone essential for regulating glucose homeostasis. It has been known for over 100 years, but its production and purification methods are still under improvement. Escherichia coli based bacterial expression system is primarily used for insulin production. The human insulin protein expressed in bacteria usually forms inclusion bodies, complicating the purification process. Traditionally, insulin purification is a timeconsuming process involving urea-based denaturation methods, and various refolding techniques, followed by extensive chromatographic methods. Here, we report an easy and efficient purification of human proinsulin involving freeze-thaw based solubilization method. The extracted proinsulin inclusion bodies are treated with different concentrations of urea, followed by a freeze-thaw based solubilization. The freezing was carried out at various temperatures, mainly -80 degrees C, -20 degrees C, and -196 degrees C to determine the optimum condition for solubilization. Highest solubilization of proinsulin from the inclusion body was achieved with 0.5M urea and -20 degrees C. Further Nickel NTA-based purification was performed, and the purified protein was characterized for disulfide mapping by high-resolution mass spectrometer (HRMS). We also performed glucose uptake assays to validate the functional properties of purified proinsulin. This freeze-thaw based mild solubilization approach is a fast and effective method for getting bioactive proinsulin, which will help further design better purification and processing strategies for insulin production.

10aDiabetes10aDisulfide mapping10aglucose10ainsulin10aMass spectrometry1 aRajesh, S.1 aJathar, Swaraj1 aBanarjee, Reema1 aSharma, Monika1 aPalkar, Shivani1 aShankar, Shiva1 aKulkarni, Mahesh, J. uhttps://library.ncl.res.in/content/simple-freeze-thaw-based-method-efficient-purification-recombinant-human-proinsulin