Proteomics > Which service should I request? > Characterization of Purified Biomolecules > Protein-metal complexes

Protein-metal complexes (e.g. metallothioneins)

General description

Protein-metal complexes can be analyzed under conditions mimicking the same environment these systems are found in nature, i.e. concentration, pH, buffer composition, ion strength. This allows to preserve the protein-metal complex from dissociation and to reveal its composition, stoichiometry, dynamics, and stability. Normally, such analyzes are being performed in water or water-methanol solutions at pH 7.5. Ammonium acetate is used to keep the pH in the solution. We do not speak about ammonium acetate buffer, since according to the latest knowledge, a drop from pH 7.5 to pH 4.75 might occur in the gas phase.

Sample preparation

If possible, samples to be submitted in a volatile buffer (e.g. ammonium acetate) at the natural pH (normally 7.5) and at the actual concentration, i.e. undiluted. Any polymeric additives or detergents must be avoided. For new customers resp. new projects, it is advantageous to inquire about the concrete requirements prior sample submission; an appointment can be set at short notice. For air-sensitive or otherwise unstable samples, an appointment for sample delivery resp. analysis has to be made beforehand.

Gel band, beads, or tissues/cell pellets can not be processed

Sample form	Procedure	Comments
Solution	Dilution in the analysis buffer	Works only for samples submitted in a volatile buffer, e.g. water, ammonium acetate. Such samples can be merely diluted prior the analysis.
Solution	Desalting using C4/C18 ZipTip	For samples in non-volatile buffers, a desalting step at the pH 7.5 using C18 or C4 ZipTips is inevitable

Samples considerations

• Minimal recommended amount: 10 µl at the highest possible concentration (0.1-10 µg/µl)
• Diluted or pre-concentrated samples can be accepted only upon request
• Gel band, beads, or tissues/cell pellets cannot be processed

Data analysis

MassLynx Software with the MaxEnt1 algorithm.

For proteins heavier than 10 kDa or if no monoisotopic signals could be recorded, for deconvolution a resolution of the output mass 0.5 Da/channel and Uniform Gaussian Damage Model at the half height of 0.7 or 0.5 Da have to be applied.
For smaller proteins i.e. MS data with monoisotopic masses were acquired, smaller parameters have to be applied, for instance, 0.02 Da/channel resp. 0.1 Da. The output will be monoisotopic masses, too.

MassLynx™ Software (Waters)(MassLynx)

Mass spectrometers measure not the overall mass of the molecule (MW) but rather its mass over charge (m/z): the observed signal is a function of both its mass and the charge state. Since chemical or electronic noise are produced during the measurement, which makes it sometimes challenging to distinguish between the signals of protein and the noise, some methods are employed for the deconvolution (simplification, refolding) of the charge information from the uncharged protein mass. Used in our laboratory MaxEnt1 algorithm (Waters, UK) MaxEnt1.pdf is the probabilistic approach of maximum entropy, which seeks to find the most probable parent mass spectrum which would yield the observed, convoluted signal. It also has to be mentioned that independently of the size of the analyzed protein, the MS analysis is being performed in a mass range up to 8'000 Th (limited by the quadrupole), and the acquired m'z signals then deconvoluted into mass spectra. The output is the molecular mass of the uncharged protein given in Da.

Intact Mass™ Software (Protein Metrics)

The Intact Mass software is used predominantly for LC-MS data, for the analysis of multiple samples from the same batch, or for the comparisons of results from different batches. Examples of applications are the semi-quantitative analysis of protein/peptide modifications or degradation, or stability studies performed at different time points.
The software can also be used for analyses performed by direct-infusion (ESI-MS)