Uncoiling collagen: a multidimensional mass spectrometry study

Abstract

Mass spectrometry can be used to determine structural information about ions by activating precursors and analysing the resulting series of fragments. Two-dimensional Fourier transform ion cyclotron resonance mass spectrometry (2D FT-ICR MS) is a technique that correlates the mass-to-charge (m/z) ratio of fragment and precursor ions in a single spectrum. 2D FT-ICR MS records the fragmentation of all ions in a sample without the need for isolation. To analyse specific precursors, horizontal cross-sections of the spectrum (fragment ion scans) are taken, providing an alternative to conventional tandem mass spectrometry (MS/MS) experiments. In this work, 2D FT-ICR MS has been used to study the tryptic digest of type I collagen, a large protein. Fragment ion scans have been extracted from the 2D FT-ICR MS spectrum for precursor m/z ratios: 951.81, 850.41, 634.34, and 659.34, and 2D FT-ICR MS spectra are compared with a set of 1D MS/MS spectra using different fragmentation methods. The results show that two-dimensional mass spectrometry excells at MS/MS of complex mixtures, simplifying spectra by eliminating contaminant peaks, and aiding the identification of species in the sample. Currently, with desktop computers, 2D FT-ICR MS is limited by data processing power, a limitation which should be alleviated using cluster parallel computing. In order to explore 2D FT-ICR MS for collagen, with reasonable computing time, the resolution in the fragment ion dimension is limited to 256k data points (compared to 4M data points in 1D MS/MS spectra), but the vertical precursor ion dimension has 4096 lines, so the total data set is 1G data points (4 Gbytes). The fragment ion coverage obtained with a blind, unoptimized 2D FT-ICR MS experiment was lower than conventional MS/MS, but MS/MS information is obtained for all ions in the sample regardless of selection and isolation. Finally, although all 2D FT-ICR MS peak assignments were made with the aid of 1D FT-ICR MS data, these results demonstrate the promise of 2D FT-ICR MS as a technique for studying complex protein digest mixtures.

Phase relationships in two-dimensional mass spectrometry

“…is the phase of the fragment ion packets at the start of detection, ω 2 is the reduced cyclotron frequency of the fragment, and c 2 ″, c 1 ″, and c 0 ″ are constants that depend on the sweep rate and the parameters of the experiment. Equation (20) shows that fragment ion packets have a phase function that is independent of the encoding delay t 1 , which is the only change to the pulse sequence from transient to transient in the 2D mass spectrometry experiment. The comparison between Eqs.…”

“…The comparison between Eqs. (18), (19), and (20) shows that precursor and fragment ions have different phase values. In addition, sometimes the same ion m/z can be a precursor present at the start of the pulse sequence and a fragment generated from another precursor during the fragmentation period.…”

“…Denoising algorithms have been proposed in order to decrease the amount of scintillation noise in 2D mass spectra and the pulse sequence was optimized [15][16][17]. 2D MS has been applied to small molecules and bottom-up and top-down proteomics, as well as polymer analysis [18][19][20][21][22][23][24][25]. Automated peak-assignment algorithms are being developed for routine analysis [26].…”

2D FT-ICR MS of Calmodulin: A Top-Down and Bottom-Up Approach

“…The potential of 2D FT-ICR MS as an analytical method for the structural analysis of small molecules has been recently demonstrated on cholesterol, showing the possibility for the technique to differentiate various fragmentation pathways and interrogate detected species [22]. 2D IRMPD FT-ICR MS has also been used for the analysis of peptides derived from tryptic digestion of commercially available collagen, which was not readily achievable with traditional 1D MS/MS [23] and for an ECD/ IRMPD comparative study for proteomics, using digested cytochrome c as a model [24]. In this work, 2D IRMPD FT-ICR MS was used for the structural analysis of calmodulin (CaM) [25,26], in both top-down and bottom-up protein tandem mass spectrometry approaches.…”

Two‐dimensional mass spectrometry in a linear ion trap, an in silico model

“…De‐noising algorithms have been developed in order to reduce the effects of scintillation noise in 2D mass spectra . 2D MS has been successfully applied to the analysis of small molecules as well as in bottom‐up and top‐down proteomics . In 2D MS, the correlation between precursor ions and their products is fully multiplexed, which enables the differentiation, for example, of product ion patterns between two precursor ion species with different charge states but with overlapping isotopic distributions .…”