Direct Quantitation of Peptide Mixtures without Standards Using Clusters Formed by Electrospray Ionization Mass Spectrometry

Abstract

In electrospray ionization mass spectrometry, ion abundances depend on a number of different factors, including analyte surface activity, competition between analytes for charge, analyte concentration, as well as instrumental factors, including mass-dependent ion transmission and detection. Here, a novel method for obtaining quantitative information about solution-phase concentrations of peptide mixtures is described and demonstrated for five different peptide mixtures with relative concentrations ranging from 0.05% to 50%. In this method, the abundances of large clusters containing anywhere from zero to thirteen impurity molecules are measured and directly related to the relative solution-phase concentration of the peptides. For clusters containing ~15 or more peptides, the composition of the clusters approaches the statistical value indicating that these clusters are formed nonspecifically and that any differences in ion detection or ionization efficiency are negligible at these large cluster sizes. This method is accurate to within ~20% or better, even when the relative ion intensities of the protonated monomers can differ by over an order of magnitude compared to their solution-phase concentrations. Although less accurate than other quantitation methods that employ internal standards, this method does have the key advantages of speed, simplicity, and the ability to quantitate components in solution even when the identity of the components are unknown.

Standard-Free Quantitation of Mixtures Using Clusters Formed by Electrospray Mass Spectrometry

“…A newly introduced standard-free quantitation method, which uses the abundances of larger molecular clusters formed by ESI to obtain relative solution-phase molar fractions,37 was investigated using L-serine as a major component. Serine has a strong propensity to form “magic” number clusters that show either homo- or heterochiral preferences and is a rigorous test of this method, which requires that impurity molecules are incorporated statistically and do not influence the ionization/detection efficiency of larger clusters.…”

“…A new standard-free quantitation method to obtain solution molar fractions using the abundances of cluster ions formed by ESI was recently introduced 37. The composition of clusters formed from peptide-containing solutions approached that in the bulk solution with increasing cluster sizes.…”

“…The composition of clusters formed from peptide-containing solutions approached that in the bulk solution with increasing cluster sizes. From the abundances of clusters containing ~15 or more peptide molecules, the solution composition could be determined to within ~20% or better even in cases where the ionization/detection efficiency of the individual molecules differed by over an order of magnitude 37. This method has the advantages that reasonably accurate quantitative information can be obtained directly from an ESI mass spectrum without using either an internal or external standard, the components do not need to be identified, and effects of instrument or detector mass bias are significantly reduced.…”

“…A critical requirement to obtain quantitative information using this method is that clusters must form statistically, which appears to occur for large peptide clusters 37. Clusters of small molecules often exhibit “magic” numbers, such as H(H 2 O) 21 + ,38 which can either be due to the special stability of the specific cluster or instability of adjacent clusters.…”

Simultaneous Quantitation of Amino Acid Mixtures using Clustering Agents

“…For a two-component mixture, molar fractions can be obtained using either a binomial expansion or, more rigorously, a weighted average [45]; in cases where the absolute concentration of one component is known, the absolute concentration of the other component can be readily obtained. In the same way, cluster ion abundances from solutions containing more than two components can also be used to determine absolute molar fractions for each component.…”