Simon Weidmann1, Gediminas Mikutis1, Konstantin Barylyuk1, and Renato Zenobi1
- Department of Chemistry and Applied Biosciences, ETH (Swiss Federal Institute of Technology) Zürich, CH-8093 Zürich, Switzerland
In high-mass matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), the accessible m/z range is limited by the detector used. Therefore, special high-mass detectors based on ion conversion dynodes (ICDs) have been developed. Recently, we have found that mass bias may exist when such ICD detectors are used [Weidmann et al., Anal. Chem. 85(6), 3425-3432 (2013)]. In this contribution, the mass-dependent response of an ICD detector was systematically studied, the response factors for proteins with molecular weights from 35.9 to 129.9 kDa were determined, and the reasons for mass bias were identified. Compared with commonly employed microchannel plate detectors, we found that the mass discrimination is less pronounced, although ions with higher masses are weakly favored when using an ICD detector. The relative response was found to depend on the laser power used for MALDI; low-mass ions are discriminated against with higher laser power. The effect of mutual ion suppression in dependence of the proteins used and their molar ratio is shown. Mixtures consisting of protein oligomers that only differ in mass show less mass discrimination than mixtures consisting of different proteins with similar masses. Furthermore, mass discrimination increases for molar ratios far from 1. Finally, we present clear guidelines that help to choose the experimental parameters such that the response measured matches the actual molar fraction as closely as possible.
CovalX Technology Used (Click each option to learn more)
Protein samples were prepared by diluting the proteins with water and phosphate buffered saline (PBS) so that the buffer concentration was kept at a constant. The total protein concentration was kept at 0.8 μM. Samples were mixed with matrix (sinapic acid (10 mg/mL) in ACN/water/TFA (49.5/49.5/1, vol:vol:vol) using a 1:1 ratio before 0.5 μL aliquots were spotted onto a stainless steel MALDI plate. The samples were allowed to crystallize at ambient temperatures and then analyzed using a mass spectrometer that had been modified with a CovalX HM2 detection system. Through this experimentation, researchers were able to determine the relative response factors of protein oligomers and DARPins with varying masses and molar ratios. They found that relative response was influenced by laser power, sample composition and ionization efficiency of sample constituents.