Authors
Simon Weidmann1, Konstantin Barylyuk1, Nadezhda Nespovitaya1, Stefanie Mädler1, and Renato Zenobi1
Organizations
- Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093 Zurich, Switzerland
Abstract
The application of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the analysis of high-mass proteins requires suitable calibration standards at high m/z ratios. Several possible candidates were investigated, and concatenated polyproteins based on recombinantly expressed maltodextrin-binding protein (MBP) are shown here to be well-suited for this purpose. Introduction of two specific recognition sites into the primary sequence of the polyprotein allows for the selective cleavage of MBP3 into MBP and MBP2. Moreover, these MBP2 and MBP3 oligomers can be dimerized specifically, such that generation of MPB4 and MBP6 is possible as well. With the set of calibrants presented here, the m/z range of 40–400 kDa is covered. Since all calibrants consist of the same species and differ only in mass, the ionization efficiency is expected to be similar. However, equimolar mixtures of these proteins did not yield equal signal intensities on a detector specifically designed for detecting high-mass molecules.
CovalX Technology Used (Click each option to learn more)
Outcomes
A study was performed in order to determine the best way to calibrate a MALDI mass spetrometer in the high-mass range. Lyophilized rh Fn was reconstituted in PBS at a concentration of 100 μg/mL and phosphorylase B was reconstituted at a concentration of 0.5 mg/mL in water. Tg bov, GroEL and PHP were unaltered. 10 nmol MBP3 was incubated with 1 μL AcTEV protease (10 U/mL) or with 6 μL of 2 μg/mL enterokinase at 25 °C for 16-24 hours to enzymatically cleave MBP3 into MBP2 and MBP. Otherwise, MBP3 was used directly in a concentration between 1 and 5 μM. 1 μL of the samples were mixed with 1 μL of matrix (SA dissolved at 10 mg/mL in 49.5/49.5/1 acetonitrile/water/TFA (v:v:v)) before 0.5 μL to 1 μL of the mixture was spotted on a stainless steel MALDI plate and crystallized. The crystallized plate was analyzed using a mass spectrometer that had been modified with a CovalX HM2 detection system. From these results, the researchers were able to determine that Tg bov, rhFn, PHP and GroEL are not suitable for use in calibrating mass spectrometers because of their wide signals.
Source
10.1021/ac400129h