Quantification of Hemoglobin A2 by Tandem Mass Spectrometry

Daniel, Yvonne; Turner, Charles; Haynes, Roberta M.; Hunt, Beverley J.; Dalton, R. Neil

doi:10.1373/clinchem.2007.088682

Cited by 68 publications

(49 citation statements)

References 22 publications

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“…Blood spots were punched (3.2 mm) into 96 deep-well polypropylene plates (Porvair Sciences). Hb peptides were assayed by MS/MS following tryptic digestion of extracts from dried blood spots in accordance with the SpOtOn Clinical Diagnostics protocol (11,12 ). The kit contains a stable isotope HbS peptide internal standard that reveals itself only following trypsin digestion and acts as a control for the digestion step.…”

Section: Analysis Of Hb Peptides In Blood Spots By Ms/msmentioning

confidence: 99%

Newborn Blood Spot Screening for Sickle Cell Disease by Using Tandem Mass Spectrometry: Implementation of a Protocol to Identify Only the Disease States of Sickle Cell Disease

Moat¹,

Rees²,

King

et al. 2014

Clinical Chemistry

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BACKGROUND The currently recommended technologies of HPLC and isoelectric focusing for newborn blood spot screening for sickle cell disease (SCD) identify both the disease and carrier states, resulting in large numbers of infants being followed up unnecessarily. Analysis of blood spot tryptic peptides performed by using tandem mass spectrometry (MS/MS) is an alternative technology to detect hemoglobin (Hb) variant disorders. METHODS We analyzed 2154 residual newborn blood spots and 675 newborn blood spots from infants with Hb variants by using MS/MS after trypsin digestion. Screening cutoffs were developed by using the ratio between the variant peptide–to–wild-type peptide abundance for HbS, C, DPunjab, OArab, Lepore, and E peptides. A postanalytical data analysis protocol was developed using these cutoffs to detect only the disease states of SCD and not to identify carrier states. A parallel study of 13 249 newborn blood spots from a high-prevalence SCD area were analyzed by both MS/MS and HPLC. RESULTS Screening cutoffs developed distinguished the infants with the disease states of SCD, infants who were carriers of SCD, and infants with normal Hb. In the parallel study no false-negative results were identified, and all clinically relevant cases were correctly identified using the MS/MS protocol. Unblinding the data revealed a total of 328 carrier infants that were successfully excluded by the protocol. CONCLUSIONS The screening protocol developed correctly identified infants with the disease states of SCD. Furthermore, large numbers of sickle cell carrier infants were successfully not identified, thereby avoiding unnecessary follow-up testing and referral for genetic counseling.

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Section: Analysis Of Hb Peptides In Blood Spots By Ms/msmentioning

confidence: 99%

Newborn Blood Spot Screening for Sickle Cell Disease by Using Tandem Mass Spectrometry: Implementation of a Protocol to Identify Only the Disease States of Sickle Cell Disease

Moat¹,

Rees²,

King

et al. 2014

Clinical Chemistry

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“…Mass spectrometry-based approaches for identifying hemoglobin variants have been developed both for whole blood and for samples resolubilized from dried blood spots [15][16][17][18][19][20][21]. Typically, mass analysis of tryptic peptides of the hemoglobin is performed.…”

Section: Introductionmentioning

confidence: 99%

“…That is, a bottom-up approach is utilized. For example, Daniel et al developed a multiple reaction monitoring method for the analysis of tryptic digests of whole blood and latterly to blood eluted from dried blood spots [15,18]. An alternative approach involves mass analysis of intact globin chains followed by MS/MS of tryptic peptides [20,21] .…”

Section: Introductionmentioning

confidence: 99%

Top-Down Proteomics and Direct Surface Sampling of Neonatal Dried Blood Spots: Diagnosis of Unknown Hemoglobin Variants

Edwards

Griffiths

Bunch

et al. 2012

J. Am. Soc. Mass Spectrom.

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We have previously shown that liquid microjunction surface sampling of dried blood spots coupled with high resolution top-down mass spectrometry may be used for screening of common hemoglobin variants HbS, HbC, and HbD. In order to test the robustness of the approach, we have applied the approach to unknown hemoglobin variants. Six neonatal dried blood spot samples that had been identified as variants, but which could not be diagnosed by current screening methods, were analyzed by direct surface sampling top-down mass spectrometry. Both collision-induced dissociation and electron transfer dissociation mass spectrometry were employed. Four of the samples were identified as β-chain variants: two were heterozygous Hb D-Iran, one was heterozygous Hb Headington, and one was heterozygous Hb J-Baltimore. The fifth sample was identified as the α-chain variant heterozygous Hb Phnom Penh. Analysis of the sixth sample suggested that it did not in fact contain a variant. Adoption of the approach in the clinic would require speed in both data collection and interpretation. To address that issue, we have compared manual data analysis with freely available data analysis software (ProsightPTM). The results demonstrate the power of top-down proteomics for hemoglobin variant analysis in newborn samples.

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“…The descriptions of a number of validated assays have been published in Clinical Chemistry (5)(6)(7)(8), and some of these assays are now being offered commercially. Although these assays may not reflect the lofty expectations the proteomics research community has established in tackling the problem of discovering new biomarkers, these assays demonstrate a clear, positive progression of the technology in the clinical laboratory.…”

mentioning

confidence: 99%

A Step Toward Simplicity for a Complex Analyte

Bystrom

2011

Clinical Chemistry

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2 deficiency is a serious, lifethreatening disorder driven by abnormally low circulating concentrations of A1AT, which lead to proteaseantiprotease imbalance. This imbalance accentuates protease activity and can lead to tissue damage, the most important of which leads to impairment of lung function (1 ). The apparent simplicity of diagnosing this disorder by a single measurement of the A1AT concentration is belied by the variety of A1AT tests offered by every reference laboratory. Physicians frequently evaluate genotyping, phenotyping, and concentration measurements in toto or through results produced via an algorithm that involves reflexing from one test to another (2 ). The need for this complex approach reflects the intricate relationships between genotype, phenotype, protein function, and concentration. The challenge in arriving at appropriate diagnosis and treatment comes from the variable penetrance of A1AT genetic defects and the limitations of each test. Typical genotyping tests detect only the most common disease alleles, S and Z, unless full exon sequencing is used. Phenotyping by isoelectric focusing can detect known and unknown polymorphs, but only in cases in which the amino acid substitution leads to a change in the protein's isoelectric point. Finally, A1AT is an acute-phase protein, and circulating concentrations can increase dramatically under conditions of stress, infection, and inflammation, perhaps providing transiently "normal" circulating A1AT concentrations, even in the presence of disease. Therefore, a correct diagnosis of deficiency requires that highly specific molecular information be used in concert with concentration measurements.In this issue of Clinical Chemistry, Chen et al. (3 ) describe a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach for determining both the phenotype and the A1AT concentration in a single assay, in contrast to the usual approach of running 2 independent assays involving different techniques. This LC-MS/MS method is clearly an improvement for any laboratory seeking to improve productivity. Although splitting samples is a common and often necessary activity in many laboratories, it adds costs and increases the chances for error. The mass spectrometry approach has more to offer because it takes advantage of the fact that it can provide quantitative information at a level of molecular detail that is very difficult to achieve by any other method.Determinations of A1AT phenotype have typically been performed via inspection of isoelectric focusing gels. For polymorphs leading to amino acid substitutions that change the protein's charge, differences in migration are observed. Despite improvements in gelbased approaches, which originally were used to discover the disease in 1963 (4 ), and the availability of electrophoresis test platforms cleared by the US Food and Drug Administration, phenotyping remains a subjective, interpretive exercise. Although gel-based techniques for protein separation are tremendously powerful, the observation of protein ba...

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Quantification of Hemoglobin A2 by Tandem Mass Spectrometry

Cited by 68 publications

References 22 publications

Newborn Blood Spot Screening for Sickle Cell Disease by Using Tandem Mass Spectrometry: Implementation of a Protocol to Identify Only the Disease States of Sickle Cell Disease

Newborn Blood Spot Screening for Sickle Cell Disease by Using Tandem Mass Spectrometry: Implementation of a Protocol to Identify Only the Disease States of Sickle Cell Disease

Top-Down Proteomics and Direct Surface Sampling of Neonatal Dried Blood Spots: Diagnosis of Unknown Hemoglobin Variants

A Step Toward Simplicity for a Complex Analyte

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