Top-Down Proteoform Analysis by 2D MS with Quadrupolar Detection

Abstract

Two-dimensional mass spectrometry (2D MS) is a multiplexed tandem mass spectrometry method that does not rely on ion isolation to correlate the precursor and fragment ions. On a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS), 2D MS instead uses the modulation of precursor ion radii inside the ICR cell before fragmentation and yields 2D mass spectra that show the fragmentation patterns of all the analytes. In this study, we perform 2D MS for the first time with quadrupolar detection in a dynamically harmonized ICR cell. We discuss the advantages of quadrupolar detection in 2D MS and how we adapted existing data processing techniques for accurate frequency-to-mass conversion. We apply 2D MS with quadrupolar detection to the top-down analysis of covalently labeled ubiquitin with ECD fragmentation, and we develop a workflow for label-free relative quantification of biomolecule isoforms in 2D MS.

Scheme 1. Pulse Sequence for the 2D MS Experiment with Frequency and m/z Range for Quadrupolar Detection

Dimensions are not to scale.

Figure 1

(a) 2D ECD mass spectrum of acetylated ubiquitin. An asterisk (*) indicates electron capture lines (green). (b) Zoom-in on the fragmentation pattern of [M + H]⁹⁺ with 4–6 acetylations. The red lines indicate dissociation lines for the various c and z fragments listed around the periphery.

Figure 2

(a) Extracted autocorrelation line from the 2D mass spectrum. The inset shows a zoomed-in view of the isotopic distribution of the [M + 10H + 4Ac]¹⁰⁺. The arrow marks the monoisotopic peak (MI). (b) Mass spectrum of acetylated ubiquitin. The inset shows the zoomed-in isotopic distribution of the [M + 10H + 4Ac]¹⁰⁺ species from the mass spectrum shown in Figure 2b. The arrow marks the monoisotopic peak (MI).

Figure 3

(a) Added-up fragment ion scans of m/z 1098 ([M + 8H + 5Ac]⁸⁺) extracted from the 2D mass spectrum of acetylated ubiquitin. (b) Zoom-in on the isotopic distribution of [M + 8H + 4Ac – NH₃]⁵⁺ from the [M + 8H + 5Ac]⁸⁺ precursor, with lines marking the fragment ion scans added up to obtain Figure 2a. (c) Sequence coverage of [M + 8H + 5Ac]⁸⁺, totaling 86% (acetylated residues underlined).

Figure 4

Acetylation rate vs residue index for ubiquitin modified with (a) four acetylations (c fragments on top, z fragments at the bottom), (b) five acetylations (c fragments on top, z fragments at the bottom), and (c) six acetylations (c fragments on top, z fragments at the bottom).

Figure 5

Precursor ion scans for c₃ (m/z 390.21790, blue) and c₃ + Ac (m/z 432.22714, red) fragments for (a) 10+, (b) 9+, (c) 8+, and (d) 7+ precursor charge states. An asterisk (*) indicates an artifact caused by the denoising algorithm.