Introduction
By ESI-MS, proteins and peptides in liquid phase are sprayed through a capillary at high voltage into the MS instrument where the mass over charge ratio (m/z) is measured. For large biomolecules the electrospray process results in multiply protonated molecules with a distribution of ion species at m/z range from 1-3000. The mass of the molecule is determined using a deconvolution algorithm that calculates the mass of the intact non-protonated protein or peptide. The ESI process requires that the sample preparation is quite pure without interfering salts or detergents.
The sample is purified by reversed phase HPLC (Agilent 1200 system) using a short C8 column before being analyzed on a Q-TOF mass spectrometer (Bruker Maxis Impact system). The mass of the sample is determined by deconvolution of the obtained raw spectra using the MaxEnt algorithm. The high resolution and accuracy of the Q-Tof instrument result in very accurate mass determination within 0-3 Da of the theoretical mass.
Sample Preparation Guidelines
Protein samples can be submitted in liquid or as lyophilized material.
The chromatographic protein purity should be >90%
Avoid detergents and keep buffer concentration at a minimum. LC-ESI MS analysis can be done on samples containing small amounts of salts and urea, but the best results are obtained with low buffer strength in volatile solvents without detergents keeping buffer concentration at a minimum. Detergents should be avoided if at all possible – otherwise please consult us before shipping samples so we can evaluate if the sample can be cleaned up for analysis.
Minimum amount ~ 10-50 micrograms
Put the protein/peptide into microcentrifuge tube
Use Eppendorf Safe Lock tubes, or similar tubes from your lab
Lyophilize or speed vac the protein to a solid sample to ensure protein stability during shipment
Alternatively, freeze or refrigerate to +4°C for cold shipment of the liquid sample
Attention
Please note that MW determination of intact proteins by mass spectrometry CANNOT be conducted on samples from gels or PVDF membranes!
