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Protein / peptide mass determination (ESI-MS, LC-MS or MALDI-MS)

General description

The determination of the mass of a protein/peptide is one way to confirm the identity of a protein and to provide insights into the presence of chemical / biological modifications. Examples of applications include the characterization of naturally or chemically modified proteins, the validation of sample integrity, or the assessment of sample heterogeneity. Analyses are mainly performed via electrospray ionization (ESI) and Matrix-assisted laser desorption/ionization (MALDI)-MS using routine or custom workflows. For complex protein mixtures, liquid chromatography analyses (LC-MS) allow the detection of signals not detectable by direct-infusion (ESI-MS)

Sample preparation

Different types of sample preparations can be performed depending on the type of protein and the composition of the buffer.
Gel band, beads, or tissues/cell pellets can not be processed

Sample formProcedureComments
SolutionDilution in the analysis bufferWorks only with volatile buffer (water, ammonium acetate, bicarbonate) or after an HPLC run. Samples at very high concentrations (e.g. 10 µg/µl) can be merely diluted (at least 1:100).
SolutionDesalting using C4/C18 ZipTip For a vast majority of proteins heavier than 10 kDa, C4 ZipTips are the better choice. Alternatively, C18 ZipTips can be used.
SolutionLC-MS analysis of intact proteinsSamples are diluted with 1% TFA to a concentration of 0.5-1 ug/ul. The final volume should be at least 20 µl
Solution - membrane proteinsTCA precipitation (if detergents are present) Precipitation by mixing equal volumes of sample and 10% TCA with a consequential addition of an additional aliquot of 50% TCA (same as 10%). After 10 min on ice, the turbid solution is centrifuged (10-13 krpm) for 10 min, and washed with cold acetone after each centrifugation step). The visible or – in most cases - invisible pellet is resolubilized in 10 µl HFIP and then 20 µl of 0.1% FA/(MeOH-2-PrOH) right before the MS analysis. No signals of the detergent are supposed to be detected anymore.
Solution - very heterogeneous samples MALDI-MSAnalysis via MALDI-MS (1+ and 2+ ion can better resolve the protein signals). Desalting using a SEC cartridge may be required
Dried samplesResolubilize before analysisdry HPLC fractions are resolubilized in the analysis buffer. Samples containing salts require desalting (see above)

Samples considerations

  • Minimal recommended amount: 10 µl at the highest possible concentration (0.5-10 µg/µl)
  • Exceptionally, 1-2 µl of a highly concentrated sample (limited amount) may be accepted. NOTE: at least 10 µl of water/buffer will be added to the sample for storage.
  • Diluted or pre-concentrated samples cannot be accepted.
  • Gel band, beads, or tissues/cell pellets cannot be processed

Data analysis

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)

Created by paolo. Last Modification: 2023-03-31 15:03 by chiawei.