Matrix-assisted ionization vacuum for protein detection, fragmentation and PTM analysis on a high resolution linear ion trap-orbitrap platform

Abstract

Matrix-assisted ionization vacuum (MAIV) is a novel ionization technique that generates multiply charged ions in vacuum without the use of laser ablation or high voltage. MAIV can be achieved in intermediate-vacuum and high-vacuum matrix-assisted laser desorption/ionization (MALDI) sources and electrospray ionization (ESI) sources without instrument modification. Herein, we adapt MAIV onto the MALDI-LTQ-Orbitrap XL platform for biomolecule analysis. As an attractive alternative to MALDI for in solution and in situ analysis of biomolecules, MAIV coupling to high resolution and accurate mass (HRAM) MS instrument has successfully expanded the mass detection range and improved the fragmentation efficiency due to the generation of multiply charged ions. Additionally, the softness of MAIV enables potential application in labile post-translational modification (PTM) analysis. In this study, proteins as large as 18.7 kDa were detected with up to 18 charges; intact peptides with labile PTM were well preserved during the ionization process and characterized MS/MS; peptides and proteins in complex tissue samples were detected and identified both in liquid extracts and in situ. Moreover, we demonstrated that this method facilitates MS/MS analysis with improved fragmentation efficiency compared to MALDI-MS/MS.

Novel ionization processes for use in mass spectrometry: ‘Squeezing’ nonvolatile analyte ions from crystals and droplets

“…v MAI‐MS/MS (CID) from tissues digested with trypsin provide excellent sequence coverage directly from tissue, an accomplishment, to the best of our knowledge, not readily duplicated with MALDI, ESI, or desorption‐ESI (DESI). These and similar improvements have been also reported by Li and coworkers using CID and ETD …”

“…Of course, cooling can cure these issues, but they have also been partially circumvented by the use of binary matrices, making the sample more stable, and/or enhancing the speed of acquisition . Similar observations have been described by the groups of McEwen, Li, and Caprioli . Obviously, the ‘matrix’ plays a critical role and details are expanded below.…”

“…A number of different mass spectrometers, in terms of analyzer types and vendors, have been used with the novel ionization processes demonstrating the importance of pressure, temperature, and collisions in optimizing ion formation . An example is shown in Figure for bovine serum albumin (BSA), 66 kDa, using mass spectrometers to ionize this 66 kDa protein intact and multiply charged using the new ionization processes.…”

“…51,55,67,69,70 A number of different mass spectrometers, in terms of analyzer types and vendors, have been used with the novel ionization processes demonstrating the importance of pressure, temperature, and collisions in optimizing ion formation. [88][89][90][91][92][93][94][95][96][97][98][99][100][101][103][104][105][106][107][108][109][110][111][113][114][115][116][117] An example is shown in Figure 2 for bovine serum albumin (BSA), 66 kDa, using mass spectrometers to ionize this 66 kDa protein intact and multiply charged using the new ionization processes. MAI, on a vacuum MALDI source without the use of the laser, produces +37 charges with 20 V extraction voltage applied using 3-NBN matrix with a Waters QTOF SYNAPT G2 mass spectrometer (Figure 2A).…”

“…However, it should be noted that introduction of a wet sample into an inlet tube increases the potential for carryover in subsequent acquisitions. A variety of different commercial MALDI and ESI sources have been employed including those from Thermo, Waters, Sciex, Bruker, and Advion mass spectrometers using MAI conditions …”