LC-MS/MS for "Liquid Chromatography coupled to tandem Mass Spectrometry".

Protein identification

Identification of proteins included in mixtures and coming from mono or bi-dimensional electrophoresis gels or from chromatography fractions is a classical application of LC-MS/MS. According to the availability of genomic data (proteins or ESTs according to the available data) of the studied species, the identification can be realized via two approaches:

1- The "Mass matching" approach or fragmentation fingerprint which aims at comparing the measured masses of peptide fragments with theoretical fragmentation masses obtained from a protein database. Via this approach, fragmentation data are not translated into amino acid sequences.

  • Software used : X!Tandem
  • Advantage: The approach allows an easy identification of proteins in very complex mixtures.
  • Limitation: The approach will identify a peptide only if it is really present in the database. Consequently, the peptide sequence must be strictly identical and non modified.

2- The "De Novo" approach which consists in the direct interpretation and translation of fragmentation spectra into amino acid sequences. Although the obtained sequences are incomplete, they can be uses to identify proteins by sequence homology.

Automatic interpretation of spectra

  • Softwares used: Peaks Studio or Pepnovo.
  • Limitations: Only high quality MS /MS spectra can be translated into amino acid sequences, which remain incomplete.
  • Attention : A few amino acids are isobaric or have very similar masses and cannot be distinguished by this approach: I=L, F=Mox, Q=K ...

Identification by sequence homology

  • Softwares used : Fasts or MSBLAST which allow the alignment of a group of peptides on a protein in a database.
  • Advantage: This approach gives access to the functions of proteins present in the sample, even when no sequence of the studied species is present in the databases.
  • Limitation: The analysis of complex mixtures is time consuming and difficult via this approach, which is more efficient for proteins coming from bi-dimensional gel electrophoresis. Moreover, the approach needs a significant level of expertise.

Other applications

In addition to protein identification, the platform uses LC-MS/MS for:

  • The characterization of post-translational modifications

The platform has expertise in the determination of phophorylation sites, analysis of disulfur bridges and in the characterization of N-terminal end of proteins.

  • Label-free quantification

The platform is developping this approach and already got interesting results in the framework of the dynamic analysis of partners of a protein complex.

Principle of the LC-MS/MS analysis

Peptides resulting from the enzymatic hydrolysis of proteins will be first separated by reverse phase liquid chromatography according to their hydrophobicity. Peptides will arrive sequencially in the mass spectrometer source (nanospray) and will be ionized.

Second, peptides will be analyzed in the mass spectrometer. This one is capable of measuring masses of peptides (simple MS) and to fragment these peptides (MS/MS spectra) to access to the peptide structures.

Example of an annotated MS/MS spectrum

      Example of an annotated MS/MS spectrum