Extending the Laserspray Ionization Concept to Produce Highly Charged Ions at High Vacuum on a Time-of-Flight Mass Analyzer

Abstract

A new matrix compound, 2-nitrophloroglucinol, is reported which not only produces highly charged ions similar to electrospray ionization (ESI) under atmospheric pressure (AP) and intermediate pressure (IP) laserspray ionization (LSI) conditions but also the most highly charged ions so far observed for small proteins in mass spectrometry (MS) under high vacuum (HV) conditions. This new matrix extends the compounds that can successfully be employed as matrixes with LSI, as demonstrated on an LTQ Velos (Thermo) at AP, a matrix-assisted laser desorption/ionization (MALDI)-ion mobility spectrometry (IMS) time-of-flight (TOF) SYNAPT G2 (Waters) at IP, and MALDI-TOF Ultraflex, UltrafleXtreme, and Autoflex Speed (Bruker) mass spectrometers at HV. Measurements show that stable multiple charged molecular ions of proteins are formed under all pressure conditions indicating softer ionization than MALDI, which suffers a high degree of metastable fragmentation when multiply charged ions are produced. An important analytical advantage of this new LSI matrix are the potential for high sensitivity equivalent or better than AP-LSI and vacuum MALDI and the potential for enhanced mass selected fragmentation of the abundant highly charged protein ions. A second new LSI matrix, 4,6-dinitropyrogallol, produces abundant multiply charged ions at AP but not under HV conditions. The differences in these similar compounds ability to produce multiply charged ions under HV conditions is believed to be related to their relative ability to evaporate from charged matrix/analyte clusters.

A Mechanism for Ionization of Nonvolatile Compounds in Mass Spectrometry: Considerations from MALDI and Inlet Ionization

“…Interestingly, the most volatile, 2-NPG, and least volatile, CHCA [44], of the above discussed matrices, produced the highest charge states for small proteins [45]. It is well known that CHCA produces multiply charged ions from proteins in MALDI [1], but relative to the matrix 2-NPG, the average charge state is lower and the energy imparted to the ions is higher.…”

“…Ionization of the matrix/analyte particles/droplets, and subsequent formation of the bare analyte ions, occurs at sub-ambient conditions in the heated inlet tube [23]. By employing 2-nitrophloroglucinol (2-NPG), one of several new LSI matrix compounds [44], multiple charging under IP conditions using the SYNAPT G2 MALDI source or LP conditions using MALDI-TOF mass spectrometers was recently demonstrated [45]. Charge states as high as +9 were observed for ubiquitin in a MALDI-TOF instrument and these high charge state ions are sufficiently stable to pass through the TOF reflectron.…”

“…By such mechanisms, particle or droplet contact with a surface could initiate charging ( Figure S7). However, the finding that nearly equal ion abundances and charge states are produced using different materials for inlet tube construction, and the recent demonstration that laser ablation of 2,5-DHAP [42] and 2-NPG [45] under IP conditions produce highly charged ion distributions nearly identical to those seen in AP-LSI are not easily reconciled with a purely surface charging mechanism. An alternative ionization possibility for matrix charging that can operate at AP and IP is statistical charging by an explosive fracturing mechanism, including such processes as bubble bursting.…”

“…For example, 2,5-DHB produces multiply charged ions in very low abundance under IP-LSI conditions with the SYNAPT G2 MALDI source, but 2,5-DHAP and 2-NPG matrices produce abundant highly charged ESI-like ions under vacuum conditions [42,45]. In AP-LSI, the inlet tube temperature necessary to produce multiply charged ions is 2-NPG G2,5-DHAP G2,5-DHB GCHCA, which is also the order these compounds vaporize or sublime with slowly increasing temperature at AP or prolonged exposure to vacuum conditions [44].…”

“…It is well known that CHCA produces multiply charged ions from proteins in MALDI [1], but relative to the matrix 2-NPG, the average charge state is lower and the energy imparted to the ions is higher. With CHCA, the multiply charged ions undergo metastable fragmentation in the linear flight tube making higher resolution reflectron measurements of multiply charged ions problematic [28,45,46]. On the other hand, the multiply charged ions produced with 2-NPG, as expected for ions formed by the LSI mechanism, are stable and pass through the reflectron as shown for carbonic anhydrase in Figure 4.…”