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Protein characterisation

Intact protein mass measurement

Intact proteins can be analysed by mass spectrometry either as denatured molecules or under non-denaturing conditions to preserve protein-protein interactions or other non-covalent interactions with smaller molecules such as ligands.

Denaturing intact protein mass measurement by MALDI-TOF MS: MALDI-TOF MS provides a quick, low resolution measurement of average protein masses. Common salt aducts and post-translational modifications are usually not resolved. However, it is a robust method and allows for the detection of large molecules above 100 kDa.

Denaturing intact protein mass measurement by ESI-MS: We use various workflows for the analysis of intact proteins by nano- or microflow ESI-coupled MS depending on the type of sample and the aim of analysis. We either run nonoflow C4 RPLC (C8 or C18 are possible too for small proteins) or nano/microflow direct infusion coupled to either LTQ-Orbitrap MS or QqTOF MS. Both mass spectrometry platforms are high resolution and accurate mass analysers that provide isotopic resolution of smaller proteins (up to ~40 kDa). In this mass range small protein modifications can be differentially detected. Even the difference in disulfide pattern (2 Da per disulfide bond) can be detected. The Orbitrap analyser provides better resolution and mass accuracy compared to the QqTOF whereas the latter has a higher mass range and provides better measurements for large proteins even up to 150 kDa. However, the quality of mass measurements strongly depends on the quality of the sample.

Native intact protein mass measurement: We use nano- or microflow direct infusion ESI-MS for the analysis of native proteins and their interactors. The sample is sprayed in a non-denaturing solvent such as ammonium acetate at various concentrations in water through a clean emitter tip. The successful measurement requires reasonably concentrated and clean sample and a well tuned instrument.

Characterisation of protein modifications

Many post-translational protein modifications (PTMs) can be identified by mass spectrometry. Modern high accuracy mass measurements give more confidence in the identification of these modifications. The resolution and mass accuracy of an Orbitrap instrument enables us to distinguish between phosphorylation vs sufation (delta mass of 0.0095), tri-methylation vs acetylation (delta mass of 0.036) or deamidation vs the 13C peak of the unmodified form (delta mass of 0.019) etc.

We use different strategies for the detection and enrichment of molecules containing certain PTMs such as phosphorylations.

In general a single protein species is differentially modified i.e. each modification may occur in different ratios and at different sites which complicates the analysis. The abundance of tryptic peptides containing PTMs is therefore lower than the abundance of tryptic peptides that never gets modified, thus finding a particular PTM can sometimes be challenging.

De novo sequencing

Not all organisms that are of significance for research are completely sequenced yet i.e. conventional mass spectrometry-based approaches for protein identification may fail. These approaches are based on the correlation of mass spectrometric data with predicted data from sequence databases.

Unknown proteins without sequence information in databases can also be sequenced with the mass spectrometer. In general this requires a manual interpretation of MS/MS-data. However the manual interpretation is error prone and many CID (collision induced dissociation)-spectra do not provide unambiguous sequence information. The available software tools for de novo interpretation of CID MS/MS-spectra are as error prone as manual interpretation.

Edman degradation and amino acid detection is still one of the most reliable methods for de novo sequencing of peptides/proteins. However, it is less sensitive than mass spectrometry, labour-intensive and expensive.

Chemically Assisted Fragmentation is a technique that generates high quality sequence data by MALDI-based tandem mass spectrometry. It is based on a chemical modification of the N-terminal amino group. The modification is negatively charged and suppresses b-ions upon fragmentation by PSD (Post Source Decay) or CID in positive ion mode. The fragment spectra contain only y-ions which simplifies the manual interpretation. We are using a different two step chemistry to firstly block the epsilon amino group of lysines by a guanidination (conversion of lysine to homoarginine) and secondly modify the N-terminus of tryptic peptides by a sulfonation with 4-sulfophenyl-isothiocyanate (SPITC). The method is described in Chen et al. (2004) Rapid Commun. Mass Spectrom, 18:191-198. High quality CID-spectra of SPITC-modified peptides reveal unambiguous sequence information by a clear y-ions series. This approach is faster and more sensitive than Edman sequencing.