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Analysis of the human chorionic gonadotropin protein at the intact level by HILIC-MS and comparison with RPLC-MS
- Source :
- Analytical and Bioanalytical Chemistry, Analytical and Bioanalytical Chemistry, Springer Verlag, 2020, ⟨10.1007/s00216-020-02684-8⟩
- Publication Year :
- 2020
-
Abstract
- In the present work, the human chorionic gonadotropin (hCG) hormone was characterized for the first time by hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution (HR) quadrupole/time-of-flight (qTOF) mass spectrometry (MS) at the intact level. This heterodimeric protein, consisting of two subunits (hCGα and hCGβ), possesses 8 potential glycosylation sites leading to a high number of glycoforms and has a molecular weight of about 35 kDa. The HILIC conditions optimized in a first paper but using UV detection were applied here with MS for the analysis of two hCG-based drugs, a recombinant hCG and a hCG isolated from the urine of pregnant women. An amide column (150 × 2.1 mm, 2.6 μm, 150 A), a mobile phase composed of acetonitrile and water both containing 0.1% of trifluoroacetic acid, and a temperature of 60 °C were used. The gradient was from 85 to 40% ACN in 30 min. The use of TFA that had been shown to be necessary for the separation of glycoforms caused, as expected, an ion suppression effect in MS that was partially overcome by increasing the amount of protein injected (2 μL at 1 mg mL−1) and reducing the detection m/z range (from 1500 to 300). These conditions allowed the detection of different glycoforms of hCGα. The performance of the HILIC-HRMS method was compared with that previously obtained in RPLC-HRMS in terms of the number of detected glycoforms, selectivity, and sensitivity. The complementarity and orthogonality of the HILIC and RP modes for the analysis of hCG at the intact level were demonstrated.
- Subjects :
- Glycosylation
Ion suppression in liquid chromatography–mass spectrometry
02 engineering and technology
Mass spectrometry
time-of-flight
01 natural sciences
Biochemistry
Chorionic Gonadotropin
Mass Spectrometry
Analytical Chemistry
Human chorionic gonadotropin
law.invention
intact protein
chemistry.chemical_compound
reversed-phase liquid chromatography
law
[CHIM.ANAL]Chemical Sciences/Analytical chemistry
Pregnancy
Trifluoroacetic acid
[CHIM]Chemical Sciences
Humans
ComputingMilieux_MISCELLANEOUS
Chromatography, High Pressure Liquid
Chromatography, Reverse-Phase
Chromatography
Chemistry
Hydrophilic interaction chromatography
010401 analytical chemistry
Reversed-phase chromatography
021001 nanoscience & nanotechnology
Recombinant Proteins
0104 chemical sciences
hydrophilic interaction chromatography
high resolution mass spectrometry
Glycoprotein Hormones, alpha Subunit
human Chorionic Gonadotropin
Recombinant DNA
Female
0210 nano-technology
Hydrophobic and Hydrophilic Interactions
Subjects
Details
- ISSN :
- 16182650 and 16182642
- Volume :
- 412
- Issue :
- 18
- Database :
- OpenAIRE
- Journal :
- Analytical and bioanalytical chemistry
- Accession number :
- edsair.doi.dedup.....0e666ec8185f1eedce2991913b412f27