Absolute Quantification of Nav1.5 Expression by Targeted Mass Spectrometry

Nav1.5 is the pore forming α-subunit of the cardiac voltage-gated sodium channel that initiates cardiac action potential and regulates the human heartbeat. A normal level of Nav1.5 is crucial to cardiac function and health. Over- or under-expression of Nav1.5 can cause various cardiac diseases rangi...

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Bibliographic Details
Published in:International journal of molecular sciences 2022-04, Vol.23 (8), p.4177
Main Authors: Adams, Sarah L, Chang, Ge, Fouda, Mohamed A, Kumar, Sharwan, Sun, Bingyun
Format: Article
Language:English
Subjects:
Action potential
Animals
Assaying
Biological properties
Brugada Syndrome
Calibration
Cardiomyocytes
CHO Cells
Coronary artery disease
Cricetinae
Cricetulus
Heart diseases
Heart failure
Humans
Mass Spectrometry
Mass spectroscopy
Mutation
NAV1.5 Voltage-Gated Sodium Channel - genetics
NAV1.5 Voltage-Gated Sodium Channel - metabolism
Peptides
Pore formation
Protein expression
Proteins
Quantitation
Regulation
Scientific imaging
Sodium
Sodium channels (voltage-gated)
Spectroscopy
Transmembrane domains
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Summary:Nav1.5 is the pore forming α-subunit of the cardiac voltage-gated sodium channel that initiates cardiac action potential and regulates the human heartbeat. A normal level of Nav1.5 is crucial to cardiac function and health. Over- or under-expression of Nav1.5 can cause various cardiac diseases ranging from short PR intervals to Brugada syndromes. An assay that can directly quantify the protein amount in biological samples would be a priori to accurately diagnose and treat Nav1.5-associated cardiac diseases. Due to its large size (>200 KD), multipass transmembrane domains (24 transmembrane passes), and heavy modifications, Nav1.5 poses special quantitation challenges. To date, only the relative quantities of this protein have been measured in biological samples. Here, we describe the first targeted and mass spectrometry (MS)-based quantitative assay that can provide the copy numbers of Nav1.5 in cells with a well-defined lower limit of quantification (LLOQ) and precision. Applying the developed assay, we successfully quantified transiently expressed Nav1.5 in as few as 1.5 million Chinese hamster ovary (CHO) cells. The obtained quantity was 3 ± 2 fmol on the column and 3 ± 2 × 104 copies/cell. To our knowledge, this is the first absolute quantity of Nav1.5 measured in a biological sample.
ISSN:1422-0067
1661-6596
1422-0067
DOI:10.3390/ijms23084177