Liquid Atmospheric Pressure Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Using a Commercial Ion Source and Orbitrap Mass Analyzer

Abstract

A liquid atmospheric pressure-matrix-assisted laser desorption/ionization (LAP-MALDI) method has been developed and applied to a commercial AP-MALDI source on a hybrid orbitrap mass analyzer. It is shown that electrospray ionization (ESI)-like mass spectra of a range of peptides and proteins can be acquired by LAP-MALDI mass spectrometry (MS) as previously demonstrated on a homemade LAP-MALDI-Q-TOF setup but without the need of any modification to the commercially available MS equipment used. Multiply charged peptide ions were recorded with a resolution of around 100,000 and a mass accuracy of less than 5 ppm. The higher resolution and mass accuracy of the orbitrap analyzer compared with previously employed Q-TOF instrumentation provided high confidence in bacterial proteoform and species identification by top-down protein analysis.

Figure 1

LAP-MALDI-orbitrap analysis of peptides and proteins. (a–d) Single-scan MALDI mass spectra of a peptide mixture using (a) conventional (solid-state) MALDI-orbitrap analysis (dried-droplet solid sample), (b) first LAP-MALDI-orbitrap analysis, (c) LAP-MALDI-Q-TOF analysis using the same sample as for panel b, and (d) second LAP-MALDI-orbitrap analysis using the same sample as for panel b and c. (e–h) Images of the LAP-MALDI sample (droplet diameter: ∼ 2 mm) used for panels b–d: (e) before any analysis, (f) after first LAP-MALDI-orbitrap analysis, (g) after LAP-MALDI-Q-TOF analysis, and (h) after second LAP-MALDI-orbitrap analysis. (i) LAP-MALDI-orbitrap mass spectrum of the triply protonated angiotensin I (50 fmol on target). (j) MS/MS spectrum of the precursor ion shown in panel i. (k) LAP-MALDI-orbitrap mass spectrum of a protein mixture. The inset displays the deconvoluted spectrum.

Figure 2

Features and applications of LAP-MALDI-orbitrap MS. (a and b) TIC and EIC (inset) using a peptide mixture for (a) LAP-MALDI-orbitrap analysis (EIC of m/z 432.9; angiotensin I [M+3H]³⁺) and (b) conventional (solid-state) MALDI-orbitrap analysis (EIC of m/z 1296.7; angiotensin I [M + H]⁺). (c) LAP-MALDI-orbitrap MS profile of K. Pneumoniae extract. (d) MS/MS data of the m/z 1194 proteoform detected in panel c. Inset shows the y₄₁⁴⁺ fragment ion. (e) Enlarged MS/MS spectrum of panel d, displaying the y₄₄⁴⁺ fragment ion and further strong ion signals of a quintuply charged fragment ion.