Exploring the Complementary Selectivity of Immunocapture and MS Detection for the Differentiation between hCG Isoforms in Clinically Relevant Samples

Lund, Hanne; Torsetnes, Silje Bøen; Paus, Elisabeth; Nustad, Kjell; Reubsaet, Léon; Halvorsen, Trine Grønhaug

doi:10.1021/pr900580n

Cited by 32 publications

(27 citation statements)

References 86 publications

(222 reference statements)

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“…hCG is also on the World Anti-Doping Agency (WADA) list of prohibited substances for male athletes (related to the use of anabolic steroids) 198 . Similar to proGRP, this protein has been extensively studied in our group 115,199,200 and there are several published papers describing sample preparing methods and quantification of hCG sampled on paper by DBS 199 and DMS 201 . Unlike proGRP, hCG contains several cysteines and is mostly present as a dimer (stabilized by three disulfide bonds), meaning that hCG is composed of two units (α and ß) were the α-unit is common for all glycoproteins and is not specific to hCG 202 .…”

Section: Human Chorionic Gonadotropinmentioning

confidence: 99%

“…The reaction was carried out at the given pH because the efficiency of IAC may be at its maximum between pH 7e8 [25], however, to avoid over-alkylation it was kept at pH 7.8 since over-alkylation could readily occur if the reaction is carried out over an extended period of time between pH 7.0 and 7.5 as suggested by Boja and Fales [25]. The concentrations investigated (50,100,150,200 and 300 mM IAC) were selected to be higher than what is normally performed in in-solution approaches to ensure sufficient reaction time (which is similar or highly correlates to the drying time, approximately 30 min). From the results (Fig.…”

Section: In-device Protein Alkylationmentioning

confidence: 99%

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Instant on-paper protein digestion during blood spot sampling

Skjærvø

Rosting

Halvorsen

et al. 2017

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A concept integrating sampling and protein digestion is introduced here combining fast and simple fabrication by wax printing on filter paper with trypsin immobilized polymer beads. The paper reactors showed promising results with a high degree of protein digestion within fifty minutes in model protein mixtures as well as in human blood. The model protein mixture was used for the evaluation of performance both with and without a reduction and alkylation step. The paper reactors without reduction and alkylation showed between 46% and 75% protein sequence coverage and between five and 20 high confidence peptides (one and five zero missed cleavage peptides, respectively). Compared to a conventional in-solution approach, the paper reactor showed 10% less protein sequence coverage, 29% fewer high confidence peptides and 19% fewer high confidence peptides with zero missed cleavages. Placement of the protein reduction and alkylation step (before or after protein digestion) was shown to be of low importance. The storage stability of the paper reactors with (six weeks) and without (twelve weeks) tryptic peptides was satisfactory. The ability of the paper reactors to digest complex biological samples was investigated by comparison with human whole blood samples prepared using a conventional dried blood spot (DBS) procedure with overnight digestion in non-targeted analysis. The reactors showed a comparable performance with 75 ± 25 for the protein groups compared to 76 ± 5 for the DBS samples. Additionally, 267 ± 72 and 335 ± 11 unique peptides (high confidence) were identified for on-paper digestion and DBS, respectively.

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Section: Human Chorionic Gonadotropinmentioning

confidence: 99%

Section: In-device Protein Alkylationmentioning

confidence: 99%

Instant on-paper protein digestion during blood spot sampling

Skjærvø

Rosting

Halvorsen

et al. 2017

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“…However, for irregular gestations, immunoassay results can be significantly different among the kits [28]. When different forms of a protein biomarker have different molecular weights, they can be readily differentiated by mass spectrometry, resulting in more reliable measurements [29]. MALDI-TOF MS can be used alone for quantification of a protein biomarker in uncomplex biological specimens, such as urine.…”

Section: Quantification Of Protein Biomarkers In Disease Diagnosismentioning

confidence: 99%

Advances in MALDI Mass Spectrometry in Clinical Diagnostic Applications

Wong

Poon

2013

Chemical Diagnostics

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The concept of matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) was first reported in 1985. Since then, MALDI MS technologies have been evolving, and successfully used in genome, proteome, metabolome, and clinical diagnostic research. These technologies are high-throughput and sensitive. Emerging evidence has shown that they are not only useful in qualitative and quantitative analyses of proteins, but also of other types of biomolecules, such as DNA, glycans, and metabolites. Recently, parallel fragmentation monitoring (PFM), which is a method comparable to selected reaction monitoring, has been reported. This highlights the potentials of MALDI-TOF/TOF tandem MS in quantification of metabolites. Here we critically review the applications of the major MALDI MS technologies, including MALDI-TOF MS, MALDI-TOF/TOF MS, SALDI-TOF MS, MALDI-QqQ MS, and SELDI-TOF MS, to the discovery and quantification of disease biomarkers in biological specimens, especially those in plasma/serum specimens. Using SELDI-TOF MS as an example, the presence of systemic bias in biomarker discovery studies employing MALDI-TOF MS and its possible solutions are also discussed in this chapter. The concepts of MALDI, SALDI, SELDI, and PFM are complementary to each other. Theoretically, all these technologies can be combined, leading to the next generation of the MALDI MS technologies. Real applications of MALDI MS technologies in clinical diagnostics should be forthcoming.

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“…Several improvements resulted in a quantitative method that could measure hCG at concentrations as low as 5 IU/L with the β T5 peptide for detection, which was shown to be unique to hCG β (20). A more recent study used an antibody recognizing multiple hCG isoforms for immunoextraction and signature peptides for detecting hCG β , nicked forms of hCG β , and hCG β cf, with a limit of quantification (LOQ) of 5 IU/L (21, 22). Although this method is a major step in the quantification of hCG isoforms in urine and blood, it does not distinguish between hCG and hCG0, since both isoforms are captured during the immunoextraction procedure and contain the same β T5 peptide used for quantification.…”

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confidence: 99%

Immunoextraction–Tandem Mass Spectrometry Method for Measuring Intact Human Chorionic Gonadotropin, Free β-Subunit, and β-Subunit Core Fragment in Urine

Woldemariam

Butch

2014

Clinical Chemistry

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BACKGROUND Human chorionic gonadotropin (hCG) stimulates testosterone production by the testicles. Because of the potential for abuse, hCG is banned (males only) in most sports and has been placed on the World Anti-Doping Agency list of prohibited substances. Intact hCG, free β-subunit (hCGβ), and β-subunit core fragment (hCGβcf) are the major variants or isoforms in urine. Immunoassays are used by antidoping laboratories to measure urinary hCG. Cross-reactivity with isoforms differs among immunoassays, resulting in widely varying results. We developed a sequential im-munoextraction method with LC-MS/MS detection for quantification of intact hCG, hCGβ, and hCGβcf in urine. METHODS hCG isoforms were immunoextracted with antibody-conjugated magnetic beads and digested with trypsin, and hCGβ and hCGβcf unique peptides were quantified by LC-MS/MS with the corresponding heavy peptides as internal standard. hCG isoform concentrations were determined in urine after administration of hCG, and the intact hCG results were compared to immunoassay results. RESULTS The method was linear to 20 IU/L. Total imprecision was 6.6%-13.7% (CV), recovery ranged from 91% to 109%, and the limit of quantification was 0.2 IU/L. Intact hCG predominated in the urine after administration of 2 hCG formulations. The window of detection ranged from 6 to 9 days. Mean immunoassay results were 12.4-15.5 IU/L higher than LC-MS/MS results. CONCLUSIONS The performance characteristics of the method are acceptable for measuring hCG isoforms, and the method can quantify intact hCG and hCGβ separately. The limit of quantification will allow LC-MS/MS hCG reference intervals to be established in nondoping male athletes for improved doping control.

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Exploring the Complementary Selectivity of Immunocapture and MS Detection for the Differentiation between hCG Isoforms in Clinically Relevant Samples

Cited by 32 publications

References 86 publications

Instant on-paper protein digestion during blood spot sampling

Instant on-paper protein digestion during blood spot sampling

Advances in MALDI Mass Spectrometry in Clinical Diagnostic Applications

Immunoextraction–Tandem Mass Spectrometry Method for Measuring Intact Human Chorionic Gonadotropin, Free β-Subunit, and β-Subunit Core Fragment in Urine

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