Mass spectrometry of single-stranded restriction fragments captured by an undigested complementary sequence

Abstract

In this report, we describe a simple and accurate method to analyze restriction fragments using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The two complementary strands of restriction fragments are separated through hybridization to a capture probe, which is a single-stranded undigested fragment. Using the biotin-streptavidin linkage, the hybrid is immobilized on streptavidin-coated magnetic beads. After conditioning the captured restriction fragments, they are eluted from the probe and their molecular weights are determined. The proposed method greatly improves the quality, and reduces the complexity of the mass spectrum by analyzing only one of the complementary strands of restriction fragments.

Figure 1

Schematic presentation of the proposed method to analyze a set of restriction fragments. The restriction fragments are prepared by digesting a PCR amplicon (199 bp) with restriction endonucleases as described in Materials and Methods. A single-stranded capture probe (sense strand) is prepared from an undigested amplicon, which has been biotinylated at the 3′ end (B = biotin). Without purifying the restriction fragments, the anti-sense strand (thin lines) of the restriction fragments is hybridized to the probe. After the hybridization, the capture probe–restriction fragment hybrid is immobilized on streptavidin-coated magnetic beads (SA = streptavidin). The captured fragments are conditioned by washing the beads, and subsequently eluted from the probe. This is followed by the analysis of the restriction fragments using MALDI-TOF MS.

Figure 2

Mass spectrum of restriction fragments. In the diagram above the spectrum, the upper thicker line represents a 3′ biotinylated capture probe (B = biotin) and the smaller lines below, which are labeled from I to VI, represent the anti-sense strand of restriction fragments.