Quantitative analysis of glycation patterns in human serum albumin using 16O/18O-labeling and MALDI-TOF MS

Omar S. Barnaby, Ronald Cerny, William Clarke, David S Hage

Research output: Contribution to journalArticle

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Abstract

Background: The glycation of human serum albumin (HSA) during diabetes can affect the ability of this protein to bind drugs and small solutes in blood. This study describes the use of 16O/18O-labeling and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to compare the levels of modification that occur throughout HSA under various glycation conditions in vitro. These quantitative studies build on a recent report that has identified the early and advanced glycation products that are formed on such samples of HSA. Methods: Glycated HSA samples were prepared by incubating 42g/l HSA with 0 to 15mmol/l glucose at pH 7.4 and 37°C for up to 5weeks. A control HSA sample was digested in 16O-enriched water and glycated HSA samples were digested in the presence of 18O-enriched water. These 2 types of samples were then mixed and the amounts of 16O- vs. 18O-labeled peptides were measured to determine the levels of modification that were occurring throughout HSA. Results: The largest levels of modification occurred in residues 101-119, 1-10 or 42-51, 87-100, 360-372, 521-531, and 275-286 of HSA after 2. weeks of glycation, and in residues 21-41, 1-10 or 42-51, 521-531, 82-93, and 146-160 after 5. weeks of glycation. Some of these regions contained the N-terminus, K199, K439, and K525, which have been previously identified as major glycation sites on HSA. The glycation pattern of HSA was dominated by early glycation products (e.g., fructosyl-lysine) after a reaction period of 2. weeks for mildly glycated HSA, while advanced glycation end products became more prominent at longer reaction times. Conclusions: The time course of the observed modifications indicated that the pattern of glycation products changed as HSA was incubated over longer periods of time with glucose. Several regions found to have significant levels of modification were at or near the major drug binding regions on HSA. These results explain why the interaction of some drugs with HSA has been observed to vary with the level of glycation for this protein.

Original languageEnglish (US)
Pages (from-to)1606-1615
Number of pages10
JournalClinica Chimica Acta
Volume412
Issue number17-18
DOIs
StatePublished - Aug 17 2011

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Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Serum Albumin
Labeling
Chemical analysis
Pharmaceutical Preparations
Glucose
Water
Medical problems
Drug Interactions
Ionization
Mass spectrometry
Desorption
Mass Spectrometry
Proteins
Lasers
Blood

Keywords

  • Diabetes
  • Human serum albumin
  • Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
  • Non-enzymatic glycation
  • O/O-Labeling
  • Quantitative proteomics

ASJC Scopus subject areas

  • Biochemistry
  • Clinical Biochemistry
  • Biochemistry, medical

Cite this

Quantitative analysis of glycation patterns in human serum albumin using 16O/18O-labeling and MALDI-TOF MS. / Barnaby, Omar S.; Cerny, Ronald; Clarke, William; Hage, David S.

In: Clinica Chimica Acta, Vol. 412, No. 17-18, 17.08.2011, p. 1606-1615.

Research output: Contribution to journalArticle

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abstract = "Background: The glycation of human serum albumin (HSA) during diabetes can affect the ability of this protein to bind drugs and small solutes in blood. This study describes the use of 16O/18O-labeling and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to compare the levels of modification that occur throughout HSA under various glycation conditions in vitro. These quantitative studies build on a recent report that has identified the early and advanced glycation products that are formed on such samples of HSA. Methods: Glycated HSA samples were prepared by incubating 42g/l HSA with 0 to 15mmol/l glucose at pH 7.4 and 37°C for up to 5weeks. A control HSA sample was digested in 16O-enriched water and glycated HSA samples were digested in the presence of 18O-enriched water. These 2 types of samples were then mixed and the amounts of 16O- vs. 18O-labeled peptides were measured to determine the levels of modification that were occurring throughout HSA. Results: The largest levels of modification occurred in residues 101-119, 1-10 or 42-51, 87-100, 360-372, 521-531, and 275-286 of HSA after 2. weeks of glycation, and in residues 21-41, 1-10 or 42-51, 521-531, 82-93, and 146-160 after 5. weeks of glycation. Some of these regions contained the N-terminus, K199, K439, and K525, which have been previously identified as major glycation sites on HSA. The glycation pattern of HSA was dominated by early glycation products (e.g., fructosyl-lysine) after a reaction period of 2. weeks for mildly glycated HSA, while advanced glycation end products became more prominent at longer reaction times. Conclusions: The time course of the observed modifications indicated that the pattern of glycation products changed as HSA was incubated over longer periods of time with glucose. Several regions found to have significant levels of modification were at or near the major drug binding regions on HSA. These results explain why the interaction of some drugs with HSA has been observed to vary with the level of glycation for this protein.",
keywords = "Diabetes, Human serum albumin, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, Non-enzymatic glycation, O/O-Labeling, Quantitative proteomics",
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T1 - Quantitative analysis of glycation patterns in human serum albumin using 16O/18O-labeling and MALDI-TOF MS

AU - Barnaby, Omar S.

AU - Cerny, Ronald

AU - Clarke, William

AU - Hage, David S

PY - 2011/8/17

Y1 - 2011/8/17

N2 - Background: The glycation of human serum albumin (HSA) during diabetes can affect the ability of this protein to bind drugs and small solutes in blood. This study describes the use of 16O/18O-labeling and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to compare the levels of modification that occur throughout HSA under various glycation conditions in vitro. These quantitative studies build on a recent report that has identified the early and advanced glycation products that are formed on such samples of HSA. Methods: Glycated HSA samples were prepared by incubating 42g/l HSA with 0 to 15mmol/l glucose at pH 7.4 and 37°C for up to 5weeks. A control HSA sample was digested in 16O-enriched water and glycated HSA samples were digested in the presence of 18O-enriched water. These 2 types of samples were then mixed and the amounts of 16O- vs. 18O-labeled peptides were measured to determine the levels of modification that were occurring throughout HSA. Results: The largest levels of modification occurred in residues 101-119, 1-10 or 42-51, 87-100, 360-372, 521-531, and 275-286 of HSA after 2. weeks of glycation, and in residues 21-41, 1-10 or 42-51, 521-531, 82-93, and 146-160 after 5. weeks of glycation. Some of these regions contained the N-terminus, K199, K439, and K525, which have been previously identified as major glycation sites on HSA. The glycation pattern of HSA was dominated by early glycation products (e.g., fructosyl-lysine) after a reaction period of 2. weeks for mildly glycated HSA, while advanced glycation end products became more prominent at longer reaction times. Conclusions: The time course of the observed modifications indicated that the pattern of glycation products changed as HSA was incubated over longer periods of time with glucose. Several regions found to have significant levels of modification were at or near the major drug binding regions on HSA. These results explain why the interaction of some drugs with HSA has been observed to vary with the level of glycation for this protein.

AB - Background: The glycation of human serum albumin (HSA) during diabetes can affect the ability of this protein to bind drugs and small solutes in blood. This study describes the use of 16O/18O-labeling and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to compare the levels of modification that occur throughout HSA under various glycation conditions in vitro. These quantitative studies build on a recent report that has identified the early and advanced glycation products that are formed on such samples of HSA. Methods: Glycated HSA samples were prepared by incubating 42g/l HSA with 0 to 15mmol/l glucose at pH 7.4 and 37°C for up to 5weeks. A control HSA sample was digested in 16O-enriched water and glycated HSA samples were digested in the presence of 18O-enriched water. These 2 types of samples were then mixed and the amounts of 16O- vs. 18O-labeled peptides were measured to determine the levels of modification that were occurring throughout HSA. Results: The largest levels of modification occurred in residues 101-119, 1-10 or 42-51, 87-100, 360-372, 521-531, and 275-286 of HSA after 2. weeks of glycation, and in residues 21-41, 1-10 or 42-51, 521-531, 82-93, and 146-160 after 5. weeks of glycation. Some of these regions contained the N-terminus, K199, K439, and K525, which have been previously identified as major glycation sites on HSA. The glycation pattern of HSA was dominated by early glycation products (e.g., fructosyl-lysine) after a reaction period of 2. weeks for mildly glycated HSA, while advanced glycation end products became more prominent at longer reaction times. Conclusions: The time course of the observed modifications indicated that the pattern of glycation products changed as HSA was incubated over longer periods of time with glucose. Several regions found to have significant levels of modification were at or near the major drug binding regions on HSA. These results explain why the interaction of some drugs with HSA has been observed to vary with the level of glycation for this protein.

KW - Diabetes

KW - Human serum albumin

KW - Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

KW - Non-enzymatic glycation

KW - O/O-Labeling

KW - Quantitative proteomics

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