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. (13)C labelling). Electrospray ionisation (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 analysing 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 analysing intact proteins larger than 100 kDa by MALDI-MS.
Type de document :
Article dans une revue
Journal of visualized experiments : JoVE, JoVE, 2013
Liste complète des métadonnées

http://hal.univ-grenoble-alpes.fr/hal-01322394
Contributeur : Frank Thomas <>
Soumis le : vendredi 27 mai 2016 - 10:25:11
Dernière modification le : jeudi 11 janvier 2018 - 06:15:24

Identifiants

Collections

UGA | IBS | CEA | IBS-IVC | DSV

Citation

Luca Signor, Elisabetta Boeri Erba. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometric analysis of intact proteins larger than 100 kDa.. Journal of visualized experiments : JoVE, JoVE, 2013. 〈hal-01322394〉

Partager

Métriques

Consultations de la notice

45