Preserving the Sequence of a Biopolymer’s Monomers as They Enter an Electrospray Mass Spectrometer

Abstract

This paper investigates how faithfully an electrospray mass spectrometer will report the order of monomers of a single biopolymer in the context of two sequencing strategies. We develop a simplified one-dimensional theoretical model of the dynamics of Brownian particles in the Taylor cone of an electropray source, where free monomers drift towards the apex in an elongational force gradient. The likelihood that neighboring particles will invert their order decreases near the apex because the strength of the force gradient increases. Neighboring monomers on a stretched biopolymer should be cleaved by photofragmentation within about 3 nm of the apex if they are to enter the mass spectrometer in sequence with 95 % probability under typical experimental conditions. Alternatively, if the monomers are cleaved processively at milliseconds-long intervals by an enzyme, their sequence will be faithfully reported with 95 % confidence if the enzyme is within about 117 nm of the apex.

The emerging landscape of single-molecule protein sequencing technologies

“…This opens a path to sequencing peptides one amino acid at a time. The concept makes use of nanopores to guide a protein into a linear configuration so that its monomers can be delivered into the mass spectrometer sequentially 34 .…”

Paving the way to single-molecule protein sequencing

“…This could, however, be overcome by chemically attaching a polypeptide to the N-terminus of proteins. Other approaches have been proposed, but lack experimental proof [100][101][102][103] . Sampath proposed the use of a double pore system in which two nanopores are placed in series 100 .…”

“…Stein and colleagues proposed the use of solid-state nanopores to create a renewed version of a mass spectrometer, in which the electrospray ionization, conventionally done with micrometre-sized nozzles, is initiated from a nanopore. This could potentially allow proteins to be sequenced if they are fragmented as they pass through the nanopore and individual amino acids are sequentially ionized and detected 103 . For a more detailed description of efforts in improving the sensitivity of mass spectrometry, we refer to other reviews 19,104 .…”

Nanopore ion sources deliver single amino acid and peptide ions directly into high vacuum

“…The droplets pass through an atmospheric-pressure background gas that induces a string of evaporation and Coulomb explosion cycles that ultimately release analyte ions into the gas phase for analysis [9]. The ESI process presents two fundamental challenges for single-protein analysis and sequencing: First, collisions with the background gas molecules quickly scramble the spatial ordering of amino acid ions, which must be preserved for sequencing [10]. Second, only a fraction of the analyte molecules passing through an electrospray source are actually measured, because many molecules never emerge from droplets as ions [9], and many of the ions that do emerge are lost to collisions with components of the mass spectrometer, especially the transfer capillary that bridges the atmospheric-pressure ion source and the high-vacuum region of the mass spectrometer [11, 12].…”

“…Sequencing would involve fragmenting the protein into its constituent amino acids as they transit the tip, and then identifying each one by determining its m/z in the mass spectrometer. The sequential ordering of monomers could be preserved by fragmenting the protein sufficiently close to the tip [10]. A magnetic sector mass analyzer combined with an array of single ion detectors can be used to monitor all the relevant mass channels simultaneously.…”