BCH3052 Lecture 14: Proteomics Methods
25 May 2018
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Lecture 14 – Proteomics Methods
Approaches to Protein Identification
• Mass spectrometry - Large instruments
• Ion source – introducing charge
o MALDI (matrix assisted laser-desorption isonisation)
▪ Absorption of UV radiation by chromophoric matrix and
ionization of matrix
▪ Dissociation of matrix phase
o ESI ion source (electrospray ionisatio)
• Mass filter – measures how fast it is going
• Mass analyzers
o Triple quadrupole mass spectrometer
Peptide Identification by MS – mass matching
• 2D-GE and MALDI-MS
o Peptide mass fingerprinting (PMF)
• 2D-GE and MS-MS
o MS peptide sequencing/fragment ion searching
• Proteomic approaches
• Bottom up Approach
o Take protein and digest it before putting it in MS
▪ Can lose some bits
• Top Down Approach
o Take intact protein and put it in MS → fractures in MS
▪ Less chance of losing bits
▪ Fuller coverage
Peptide Sequencing by MS – fragmentations
• Peptide mass fingerprinting (PMF)
o Larger the fragment, more it will be impede
o Smaller fragments travel faster
• Peptide mapping and mass fingerprint by MALDI-TOF
o Proteins are identified by matching a list of experimental peptide
masses with the calculated list of all peptide masses of each entry in a
proteomics databases.
o Mass mapping require a purified protein the technique is generally
used in conjunction with prior protein fractionation
• Cleave at CO-NH
• Subject it to frequencing by MS → gives sequence
• Multi-Dimensional Protein Identification Technology (MudPIT)
o Denatured protein → digested peptides → 2D chromatographic
separation of peptides (charged version
first → SCX → RP) → tandem mass
spectrometry → identify proteins in
complex
• MudPITS in Proteomics
o Application to membrane proteomics
o Post translational modifications
