Unprecedented Ionization Processes in Mass Spectrometry Provide Missing Link between ESI and MALDI

Abstract

In the field of mass spectrometry, producing intact, highly-charged protein ions from surfaces is a conundrum with significant potential payoff in application areas ranging from biomedical to clinical research. Here, we report on the ability to form intact, highly-charged protein ions on high vacuum time-of-flight mass spectrometers in the linear and reflectron modes achievable using experimental conditions that allow effective matrix removal from both the sample surfaces and from the charged clusters formed by the laser ablation event. The charge states are the highest reported on high vacuum mass spectrometers, yet they remain at only around a third of the highest charge obtained using laser ablation with a suitable matrix at atmospheric pressure. Other than physical instrument modifications, the key to forming abundant and stable highly-charged ions appears to be the volatility of the matrix used. Cumulative results suggest mechanistic links between the ionization process reported here and traditional ionization methods of electrospray ionization and matrix-assisted laser desorption/ionization.

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

“…To differentiate between the two dominating processes of pressure and temperature, here we differentiate by vacuum MAI ( v MAI) and inlet MAI ( i MAI), if the context requires. Multiply charged ions (+14 charges) could also be observed using 2‐nitrophloroglucinol (2‐NPG) as matrix and laser ablation on high‐vacuum Bruker MALDI‐TOF mass spectrometers (Figure C) . Using an inlet tube temperature of 200 °C on a commercial heated inlet tube atmospheric pressure ionization Thermo LTQ Velos mass spectrometer, BSA ions with up to +67 charges were observed employing 2‐NPG matrix (Figure D) .…”

“…Especially with less volatile matrices and high‐mass analytes, collisions with, for example, gases (e.g., above atmospheric pressure or pressure differentials within the instrument) have been shown to be beneficial, similar to what has been used in ESI . While ionizing voltages are not needed, some voltage can be applied with any of the methods …”

“…Using a temperature range from 70 to 400 °C on an Orbitrap Q‐Exactive Focus mass spectrometer produces for 3‐NBN matrix exceptionally high analyte ion abundance without the need for high voltages or a laser , while other matrices, especially typical MALDI matrices, produce less abundant analyte ions, multiply and singly charged, and only at high temperatures. The temperature can be applied directly or indirectly to the tube, typically used as an ESI inlet . Charge separation occurs after the matrix is introduced to sub‐atmospheric pressure.…”

“…Consequently, using mass spectrometers optimized for ESI‐ and MALDI‐MS, the ease of adaptation of the methods based on the new ionization processes varies. It may only require turning off the high voltages and/or laser, or it may necessitate more significant physical modification(s) to obtain analytical useful results . As an example, the currently best performing MAI matrix, 3‐NBN, on Waters SYNAPT G2 and G2S instruments works exceptionally well for analyte ionization on the intermediate pressure MALDI sources without the use of the laser, but performs best at room temperature on the ESI Z‐Spray source blocks, and above the melting point of the matrix, 114–117 °C, the analyte ion abundance essentially ceases on this instrument .…”

“…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.…”