Absolute Quantification of Nav1.5 Expression by Targeted Mass Spectrometry

Abstract

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.

Keywords: absolute protein quantification; cardiac diseases; cardiomyocytes; ion channels; membrane protein expression; parallel reaction monitoring (PRM); protein copy numbers; sodium voltage-gated channel alpha subunit 5 gene (SCN5A) and its corresponding protein (Nav1.5); targeted mass spectrometry.

Figure 1

Illustrations of cardiac action potential.

Figure 2

SDS-PAGE gel results of SCN5A transfected CHO cell lysates pre and post-trypsin digestion. Lane 1, pre-digestion; lane 2, 24-h post-digestion; lane 3, 48-h post-digestion.

Figure 3

MS2 fragmentation patterns of Nav1.5 peptides (Heavy form). Red color denotes amino-terminal fragments, and blue color denotes carboxy-terminal fragments.

Figure 4

Skyline extracted chromatograms of heavy and light peptides of Nav1.5, the example overlap chromatograms of a particular transition, and their corresponding calibration curves. Linear least squares regression analysis was used to obtain the fitted calibration curve, and the dot is the mean and the error bar is the standard derivation of 3 replicates.

Figure 5

Slope variations among different transitions of VLLEYADK. The results are plotted as mean ± standard deviation, and the number of repeats is three. One-way ANOVA was performed for significant differences, and p value was greater than 0.05.