Rapid determination of transferrin isoforms by immunoaffinity liquid chromatography and electrospray mass spectrometry

J. M. Lacey, Harold Robert (Bob) III Bergen, M. J. Magera, S. Naylor, J. F. O'Brien

Research output: Contribution to journalArticle

149 Citations (Scopus)

Abstract

Background: Congenital disorders of glycosylation (CDG) are autosomal recessive disorders that produce increased serum carbohydrate-deficient transferrin (CDT) isoforms. Methods to resolve CDT from fully glycosylated transferrin (Trf) have been based on a neutral shift in the isoelectric focusing (IEF) pattern or on a reduction in the negative charge, allowing resolution by anion-exchange chromatography. Our purpose was to develop a method of resolution and relative quantification of Trf isoforms using online immunoaffinity liquid chromatography-mass spectrometry (LC-MS). Methods: Serum (25 μL) was diluted with 100 μL of water before application to an immunoaffinity column that sequestered Trf isoforms. Trf isoforms were eluted from the immunoaffinity column, concentrated on a C4 column, eluted from the C4 column, and introduced into the mass spectrometer. Analysis of the Trf isoforms was entirely automated and completed in <10 min per sample. Results: The LC-MS method demonstrated that the major abnormal Trf isoforms in CDG lack one complete oligosaccharide structure (mono-oligosaccharide) or both oligosaccharide structures (a-oligosaccharide), but not the sialic acids, as presumed on the basis of IEF methods. Calculation of relative ratios among three possible species (mono-/di-oligosaccharide and a-/di-oligosaccharide) is reproducible [mean intra- and inter-assay CVs were 9.3% (n = 10) and 10% (n = 5), respectively]. A reference range for patients <18 years was determined by analysis of 209 samples (for mono-/di-oligosaccharide, the median was 0.041 and the range was 0.018-0.083; for a-/di-oligosaccharide, the median was 0.007 and the range was 0.002-0.036). Comparison of data obtained with an affinity chromatography-IEF method and the LC-MS method demonstrated equivalence in the interpreted results (n = 170). Conclusions: Advantages of the LC-MS method include improved sensitivity, minimal sample preparation, and an analysis time of <10 min. The method was automated, which allowed high throughput, with >100 samples analyzed in a single day. Moreover, the nature of the oligosaccharide defect in CDG is accurately reflected by mass resolution, and subtle oligosaccharide truncations may also be detected by this method.

Original languageEnglish (US)
Pages (from-to)513-518
Number of pages6
JournalClinical Chemistry
Volume47
Issue number3
StatePublished - 2001

Fingerprint

Liquid chromatography
Transferrin
Liquid Chromatography
Mass spectrometry
Mass Spectrometry
Protein Isoforms
Congenital Disorders of Glycosylation
Glycosylation
Oligosaccharides
Isoelectric Focusing
Mass spectrometers
Chromatography
Serum
Anions
Defects
Water
carbohydrate-deficient transferrin

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

Rapid determination of transferrin isoforms by immunoaffinity liquid chromatography and electrospray mass spectrometry. / Lacey, J. M.; Bergen, Harold Robert (Bob) III; Magera, M. J.; Naylor, S.; O'Brien, J. F.

In: Clinical Chemistry, Vol. 47, No. 3, 2001, p. 513-518.

Research output: Contribution to journalArticle

@article{cb95e8bd0ab24aada19251b95d532df1,
title = "Rapid determination of transferrin isoforms by immunoaffinity liquid chromatography and electrospray mass spectrometry",
abstract = "Background: Congenital disorders of glycosylation (CDG) are autosomal recessive disorders that produce increased serum carbohydrate-deficient transferrin (CDT) isoforms. Methods to resolve CDT from fully glycosylated transferrin (Trf) have been based on a neutral shift in the isoelectric focusing (IEF) pattern or on a reduction in the negative charge, allowing resolution by anion-exchange chromatography. Our purpose was to develop a method of resolution and relative quantification of Trf isoforms using online immunoaffinity liquid chromatography-mass spectrometry (LC-MS). Methods: Serum (25 μL) was diluted with 100 μL of water before application to an immunoaffinity column that sequestered Trf isoforms. Trf isoforms were eluted from the immunoaffinity column, concentrated on a C4 column, eluted from the C4 column, and introduced into the mass spectrometer. Analysis of the Trf isoforms was entirely automated and completed in <10 min per sample. Results: The LC-MS method demonstrated that the major abnormal Trf isoforms in CDG lack one complete oligosaccharide structure (mono-oligosaccharide) or both oligosaccharide structures (a-oligosaccharide), but not the sialic acids, as presumed on the basis of IEF methods. Calculation of relative ratios among three possible species (mono-/di-oligosaccharide and a-/di-oligosaccharide) is reproducible [mean intra- and inter-assay CVs were 9.3{\%} (n = 10) and 10{\%} (n = 5), respectively]. A reference range for patients <18 years was determined by analysis of 209 samples (for mono-/di-oligosaccharide, the median was 0.041 and the range was 0.018-0.083; for a-/di-oligosaccharide, the median was 0.007 and the range was 0.002-0.036). Comparison of data obtained with an affinity chromatography-IEF method and the LC-MS method demonstrated equivalence in the interpreted results (n = 170). Conclusions: Advantages of the LC-MS method include improved sensitivity, minimal sample preparation, and an analysis time of <10 min. The method was automated, which allowed high throughput, with >100 samples analyzed in a single day. Moreover, the nature of the oligosaccharide defect in CDG is accurately reflected by mass resolution, and subtle oligosaccharide truncations may also be detected by this method.",
author = "Lacey, {J. M.} and Bergen, {Harold Robert (Bob) III} and Magera, {M. J.} and S. Naylor and O'Brien, {J. F.}",
year = "2001",
language = "English (US)",
volume = "47",
pages = "513--518",
journal = "Clinical Chemistry",
issn = "0009-9147",
publisher = "American Association for Clinical Chemistry Inc.",
number = "3",

}

TY - JOUR

T1 - Rapid determination of transferrin isoforms by immunoaffinity liquid chromatography and electrospray mass spectrometry

AU - Lacey, J. M.

AU - Bergen, Harold Robert (Bob) III

AU - Magera, M. J.

AU - Naylor, S.

AU - O'Brien, J. F.

PY - 2001

Y1 - 2001

N2 - Background: Congenital disorders of glycosylation (CDG) are autosomal recessive disorders that produce increased serum carbohydrate-deficient transferrin (CDT) isoforms. Methods to resolve CDT from fully glycosylated transferrin (Trf) have been based on a neutral shift in the isoelectric focusing (IEF) pattern or on a reduction in the negative charge, allowing resolution by anion-exchange chromatography. Our purpose was to develop a method of resolution and relative quantification of Trf isoforms using online immunoaffinity liquid chromatography-mass spectrometry (LC-MS). Methods: Serum (25 μL) was diluted with 100 μL of water before application to an immunoaffinity column that sequestered Trf isoforms. Trf isoforms were eluted from the immunoaffinity column, concentrated on a C4 column, eluted from the C4 column, and introduced into the mass spectrometer. Analysis of the Trf isoforms was entirely automated and completed in <10 min per sample. Results: The LC-MS method demonstrated that the major abnormal Trf isoforms in CDG lack one complete oligosaccharide structure (mono-oligosaccharide) or both oligosaccharide structures (a-oligosaccharide), but not the sialic acids, as presumed on the basis of IEF methods. Calculation of relative ratios among three possible species (mono-/di-oligosaccharide and a-/di-oligosaccharide) is reproducible [mean intra- and inter-assay CVs were 9.3% (n = 10) and 10% (n = 5), respectively]. A reference range for patients <18 years was determined by analysis of 209 samples (for mono-/di-oligosaccharide, the median was 0.041 and the range was 0.018-0.083; for a-/di-oligosaccharide, the median was 0.007 and the range was 0.002-0.036). Comparison of data obtained with an affinity chromatography-IEF method and the LC-MS method demonstrated equivalence in the interpreted results (n = 170). Conclusions: Advantages of the LC-MS method include improved sensitivity, minimal sample preparation, and an analysis time of <10 min. The method was automated, which allowed high throughput, with >100 samples analyzed in a single day. Moreover, the nature of the oligosaccharide defect in CDG is accurately reflected by mass resolution, and subtle oligosaccharide truncations may also be detected by this method.

AB - Background: Congenital disorders of glycosylation (CDG) are autosomal recessive disorders that produce increased serum carbohydrate-deficient transferrin (CDT) isoforms. Methods to resolve CDT from fully glycosylated transferrin (Trf) have been based on a neutral shift in the isoelectric focusing (IEF) pattern or on a reduction in the negative charge, allowing resolution by anion-exchange chromatography. Our purpose was to develop a method of resolution and relative quantification of Trf isoforms using online immunoaffinity liquid chromatography-mass spectrometry (LC-MS). Methods: Serum (25 μL) was diluted with 100 μL of water before application to an immunoaffinity column that sequestered Trf isoforms. Trf isoforms were eluted from the immunoaffinity column, concentrated on a C4 column, eluted from the C4 column, and introduced into the mass spectrometer. Analysis of the Trf isoforms was entirely automated and completed in <10 min per sample. Results: The LC-MS method demonstrated that the major abnormal Trf isoforms in CDG lack one complete oligosaccharide structure (mono-oligosaccharide) or both oligosaccharide structures (a-oligosaccharide), but not the sialic acids, as presumed on the basis of IEF methods. Calculation of relative ratios among three possible species (mono-/di-oligosaccharide and a-/di-oligosaccharide) is reproducible [mean intra- and inter-assay CVs were 9.3% (n = 10) and 10% (n = 5), respectively]. A reference range for patients <18 years was determined by analysis of 209 samples (for mono-/di-oligosaccharide, the median was 0.041 and the range was 0.018-0.083; for a-/di-oligosaccharide, the median was 0.007 and the range was 0.002-0.036). Comparison of data obtained with an affinity chromatography-IEF method and the LC-MS method demonstrated equivalence in the interpreted results (n = 170). Conclusions: Advantages of the LC-MS method include improved sensitivity, minimal sample preparation, and an analysis time of <10 min. The method was automated, which allowed high throughput, with >100 samples analyzed in a single day. Moreover, the nature of the oligosaccharide defect in CDG is accurately reflected by mass resolution, and subtle oligosaccharide truncations may also be detected by this method.

UR - http://www.scopus.com/inward/record.url?scp=0035107128&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035107128&partnerID=8YFLogxK

M3 - Article

C2 - 11238305

AN - SCOPUS:0035107128

VL - 47

SP - 513

EP - 518

JO - Clinical Chemistry

JF - Clinical Chemistry

SN - 0009-9147

IS - 3

ER -