Front-End Electron Transfer Dissociation Coupled to a 21 Tesla FT-ICR Mass Spectrometer for Intact Protein Sequence Analysis

Abstract

High resolution mass spectrometry is a key technology for in-depth protein characterization. High-field Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) enables high-level interrogation of intact proteins in the most detail to date. However, an appropriate complement of fragmentation technologies must be paired with FTMS to provide comprehensive sequence coverage, as well as characterization of sequence variants, and post-translational modifications. Here we describe the integration of front-end electron transfer dissociation (FETD) with a custom-built 21 tesla FT-ICR mass spectrometer, which yields unprecedented sequence coverage for proteins ranging from 2.8 to 29 kDa, without the need for extensive spectral averaging (e.g., ~60% sequence coverage for apo-myoglobin with four averaged acquisitions). The system is equipped with a multipole storage device separate from the ETD reaction device, which allows accumulation of multiple ETD fragment ion fills. Consequently, an optimally large product ion population is accumulated prior to transfer to the ICR cell for mass analysis, which improves mass spectral signal-to-noise ratio, dynamic range, and scan rate. We find a linear relationship between protein molecular weight and minimum number of ETD reaction fills to achieve optimum sequence coverage, thereby enabling more efficient use of instrument data acquisition time. Finally, real-time scaling of the number of ETD reactions fills during method-based acquisition is shown, and the implications for LC-MS/MS top-down analysis are discussed. Graphical Abstract ᅟ.

Keywords: ETD; Electrospray ionization; FTMS; Fourier transform mass spectrometry; Ion-ion reaction; MS/MS; MS2; Top-down.

Figure 1

(a) Conceptual layout for the 21 T FT-ICR mass spectrometer with which all experiments were conducted. This system features a dual cell linear ion trap, a multipole storage device, a dynamically harmonized cell, and a 21 tesla actively shielded superconducting magnet. (b) Experimental event sequence, showing how multiple fill experiments were conducted

Figure 2

Fractional sequence coverage versus cumulative ion target (1 ETD fragment ion fill = 1E5 analyte charges) melittin (a), ubiquitin (b), cytochrome c (c), hemoglobin subunit (d), apo-myoglobin (e), histone H1 (f), and carbonic anhydrase (g). Low MW proteins, with fewer possible dissociation channels, require fewer fills to achieve reasonable coverage than do their larger counterparts

Figure 3

Minimum precursor AGC cumulative ion target required for optimized sequence coverage. The highly linear relationship (R² = 0.988) evidently results from a linear increase in number of dissociation channels with increasing MW

Figure 4

Scaling of the number of fills as a function of molecular weight during method-based operation of the mass spectrometer. Bottom: Positive ESI mass spectrum of a mixture of three proteins (melittin, ubiquitin, cytochrome c). The ETD product ion spectra of melittin [M + 5H]⁵⁺ was acquired with three fills (left), ubiquitin [M + 10H]¹⁰⁺ with eight fills (right), and cytochrome c [M + 16H]¹⁶⁺ with 11 fills (top). All values were determined on-the-fly during method-based acquisition from the linear expression shown in Figure 3. That equation was incorporated directly into the ITCL programming of the system

Figure 5

Fractional sequence coverage for carbonic anhydrase as a function of the number of averaged spectra. These data were acquired over a range of resolving power (m/z 400) settings from 37.5 to 600 k

Figure 6

(a) Fragment ion map generated from carbonic anhydrase [M + 34H]³⁴⁺ illustrating 87% sequence coverage from ETD fragmentation alone. The signal was averaged over 1500 acquisitions by use of 16 fills with a reaction period of 6.0 ms (RP at m/z 400 = 600,000) (b) Mass spectrum of the underlying data. Insets include 5, 50, and 100 Da ranges expanded to illustrate the spectral complexity and dynamic range within the spectrum. Large fragment ions are observed at m/z > 1800