Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Abstract

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass determination allows one to evaluate the correctness of protein primary sequences, the presence of mutations and/or post-translational modifications, the possible protein degradation, the sample homogeneity, and the degree of isotope incorporation in case of labelling (e.g. C labelling).Electrospray ionization (ESI) mass spectrometry (MS) is widely used for mass determination of denatured proteins, but its efficiency is affected by the composition of the sample buffer. In particular, the presence of salts, detergents, and contaminants severely undermines the effectiveness of protein analysis by ESI-MS. Matrix-assisted laser desorption/ionization (MALDI) MS is an attractive alternative, due to its salt tolerance and the simplicity of data acquisition and interpretation. Moreover, the mass determination of large heterogeneous proteins (bigger than 100 kDa) is easier by MALDI-MS due to the absence of overlapping high charge state distributions which are present in ESI spectra.Here we present an accessible approach for analyzing proteins larger than 100 kDa by MALDI-time of flight (TOF). We illustrate the advantages of using a mixture of two matrices (i.e. 2,5-dihydroxybenzoic acid and α-cyano-4-hydroxycinnamic acid) and the utility of the thin layer method as approach for sample deposition. We also discuss the critical role of the matrix and solvent purity, of the standards used for calibration, of the laser energy, and of the acquisition time. Overall, we provide information necessary to a novice for analyzing intact proteins larger than 100 kDa by MALDI-MS.

Current Trends in the Analysis of Post-translational Modifications

“…Analysis of proteins requires soft ionization techniques, such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), that are able to ionize thermolabile and non-volatile compounds without considerable fragmentation [24]. In MALDI, samples are co-crystallized with a large amount of UV-absorbing matrix material on a metal plate and are bombarded by a pulsed UV laser, generating volatilized and typically +1 charged particles [25]. However, novel matrices support the formation of multiply charged ions providing improved fragmentation efficiency and enabling the use of electron-based fragmentation methods, which can be highly beneficial in the analysis of labile PTMs (see later in the section) [26].…”

“…This ionization technique allows direct analysis of mixtures and biomolecules in a wide molecular mass range (~ 300-200,000 Da). Additionally, it is considerably tolerant of salt contamination [25]. The most important disadvantage of MALDI is that it is not straightforward to couple it to liquid chromatography on-line.…”

Combining a NHS ester and glutaraldehyde improves crosslinking prior to MALDI MS analysis of intact protein complexes

“…The samples were diluted (1:3, 1:4, 1:6 and 1:12) in 5% formic acid, deposited on the MALDI target and analyzed by MALDI-TOF as described previously. [31] When we used both XLers, XLing 'reaction 1' was always performed in a thermomixer for 60 min at 25°C and 300 rpm. 'Reaction 2' was either carried out in a thermomixer or using ultracentrifugation ( Supplementary Fig.…”

“…Afterwards, the samples were diluted (1:3, 1:4, 1:6 and 1:12) in 5% formic acid and analyzed by MALDI-TOF. [31] Mass spectrometry and data analysis…”

“…A mix of α-cyano-4-hydroxycinnamic acid (α-CHCA) and 2,5-dihydroxybenzoic acid (DHB) (both from Sigma-Aldrich, Saint-Quentin Fallavier, France) was used. [31] A Nd:YAG laser (355 nm) was used at the minimum intensity necessary to generate ions. Each mass spectrum was obtained by averaging 2000 laser shots fired randomly at a sample spot.…”