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Quantitative analysis of glycation sites on human serum albumin using (16)O/(18)O-labeling and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

ABSTRACT: One of the long term complications of diabetes is the non-enzymatic addition of glucose to proteins in blood, such as human serum albumin (HSA), which leads to the formation of an Amadori product and advanced glycation end products (AGEs). This study uses (16)O/(18)O-labeling and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to provide quantitative data on the extent of modification that occurs in the presence of glucose at various regions in the structure of minimally glycated HSA.Normal HSA, with no significant levels of glycation, was digested by various proteolytic enzymes in the presence of water, while a similar sample containing in vitro glycated HSA was digested in (18)O-enriched water. These samples were then mixed and the (16)O/(18)O ratios were measured for peptides in each digest. The values obtained for the (16)O/(18)O ratios of the detected peptides for the mixed sample were used to determine the degree of modification that occurred in various regions of glycated HSA.Peptides containing arginines 114, 81, or 218 and lysines 413, 432, 159, 212, or 323 were found to have (16)O/(18)O ratios greater than a cut off value of 2.0 (i.e., a cut off value based on results noted when using only normal HSA as a reference). A qualitative comparison of the (16)O- and (18)O-labeled digests indicated that lysines 525 and 439 also had significant degrees of modification. The modifications that occurred at these sites were variations of fructosyl-lysine and AGEs which included 1-alkyl-2-formyl-3,4-glycoyl-pyrole and pyrraline.Peptides containing arginine 218 and lysines 212, 413, 432, and 439 contained high levels of modification and are also present near the major drug binding sites on HSA. This result is clinically relevant because it suggests the glycation of HSA may alter its ability to bind various drugs and small solutes in blood.

SUBMITTER: Barnaby OS

PROVIDER: S-EPMC2883623 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Quantitative analysis of glycation sites on human serum albumin using (16)O/(18)O-labeling and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Barnaby Omar S OS Wa Chunling C Cerny Ronald L RL Clarke William W Hage David S DS

Clinica chimica acta; international journal of clinical chemistry 20100413 15-16

<h4>Background</h4>One of the long term complications of diabetes is the non-enzymatic addition of glucose to proteins in blood, such as human serum albumin (HSA), which leads to the formation of an Amadori product and advanced glycation end products (AGEs). This study uses (16)O/(18)O-labeling and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to provide quantitative data on the extent of modification that occurs in the presence of glucose at various ...[more]

PMID: 20394739

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Project description:Streptococcus suis, particularly S. suis serotype 2 (SS2), is an important zoonotic pathogen causing meningitis in humans worldwide. Although the proper classification of the causative and pathogenic serotype is salutary for the clinical diagnosis, cross-reactions leading to the indistinguishability of serotypes by the current serotyping methods are significant limitations. In the present study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of extracted peptides was developed to improve the classification of serotype of S. suis. The peptide mass fingerprint (PMFs) database of S. suis was generated from the whole-cell peptides of 32 reference strains of S. suis isolates obtained from pigs. Thirty-two human S. suis isolates from clinical cases in Thailand were used to validate this alternative serotyping method in direct comparison to the multiplex (m)PCR approach. All reference strains, representing 32 serotypes of S. suis, exhibited their individual PMFs patterns, thus allowing differentiation from one another. Highly pathogenic SS2 and SS14 were clearly differentiated from the otherwise serologically closely related SS1/2 and SS1, respectively. The developed MALDI-TOF-MS serotyping method correctly classified the serotype in 68.8% (22/32) of the same serotype isolates generated from the PMFs database; while the validity for the clinical human isolates was 62.5% (20/32). The agreement between the MALDI-TOF-MS and mPCR serotyping was moderate with a Kappa score of 0.522, considering that mPCR could correctly serotype up to 75%. The present study demonstrated that PMFs from the developed MALDI-TOF-MS-based method could successfully discriminate the previously indistinguishable highly pathogenic SS2 and SS14 from SS1/2 and SS1, respectively. Moreover, this serotyping method distinguished pathogenic SS6, and so is an alternative approach of choice to rapidly and reliably serotype clinically pathogenic S. suis isolates.

Dataset Information

Quantitative analysis of glycation sites on human serum albumin using (16)O/(18)O-labeling and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Publications

Quantitative analysis of glycation sites on human serum albumin using (16)O/(18)O-labeling and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

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