Improving limits of detection for B-type natriuretic peptide using PC-IDMS: an application of the ALiPHAT strategy

Abstract

Hydrophobic tagging of biomolecules has been reported by our group and others to increase their ionization efficiency during electrospray ionization and facilitate their detection by mass spectrometry. As such, hydrophobic tagging should provide a viable method for augmenting MS-based quantification of low abundance proteins by decreasing their detection limits. Herein we have evaluated two commercial alkylation reagents and several newly synthesized hydrophobic alkylation reagents for their utility in quantifying B-type Natriuretic Peptide, a low abundance cardiac biomarker, by protein cleavage isotope dilution mass spectrometry. For the cysteine containing tryptic peptide evaluated, a approximately 3.5-fold decrease in the detection limit was observed for the best performing hydrophobic reagent, 2-iodo-N-octylacetamide, relative to the commonly used alkylation reagent, iodoacetamide. Additionally, we have evaluated the use of nonpolar surface areas as a metric for assessing the effectiveness of the alkylation reagents in improving ESI response.

Figure 1

The above chromatograms were obtained during nanoLC-SRM analysis of the modified peptides. The transitions are for the natural version of the modified peptides with 8 femtomoles loaded on column. Shown in parentheses are the relative abundances as measured by the peak area. The DCAM modified peptide was magnified for visualization.

Figure 2

(A) Plotted are the average peak areas (n=3) for the natural version of the modified peptides loaded on column in different amounts. (B) The average abundances of each modified peptide are normalized to that of the IAM-modified peptide (A_X/A_IAM) to determine the fold improvement.

Figure 3

Calibration curves for each modified peptide are shown. The slopes for the displayed curves ranged between 0.9584 and 1.0787, while the intercepts were between -0.0523 to -0.0202.

Figure 4

The LOD for each modified peptide is displayed in the bar graph as attomoles of modified peptide loaded on column. To determine the fold improvements, the LOD for each species was normalized to the IAM-modified peptide (LOD_IAM/LOD_X). In terms of the total amount of material loaded on column, the LODs for IAM, NEM, OCAM, PH1, PH2, PH4, and BzAM modified peptides were 1260, 457, 357, 420, 603, 549, and 601 attomoles, respectively. Since DCAM did not provide a linear calibration curve, its detection limit could not be reliably calculated.

Figure 5

Plotted are the average peak areas (n=18) for the SIL version of the modified peptides with 8 femtomoles loaded on column versus their respective NPSAs. The NPSA for each modified peptide was estimated by summing the NPSA for the tryptic peptide and the respective reagent. Highlighted is the NPSA range that appears to provide the optimal ESI response for the BNP tryptic peptide.