Protein charecterisation and primary structure

how do you find relative motility?

distance migrated by protein / length of gel

how do you find number of identical subunits in protein?

native mwt / denatured mwt

describes steps in finding native molecular weight

1. gel filtration

2. run mix of standard proteins of known molecular weight

3. produce standard curve to estimate unknown native mwt’s

describes steps in finding denatured molecular weight

1. run SDS PAGE

2. run mix of standard denatured proteins with known mwt’s

3. produce standard curve, log mwt against relative motility

what are the 2 methods of analysing primary protein structure using mass spec

peptide mass fingerprinting (PMF)

peptide sequencing (tandem MS)

what is the basic rule of PMF

proteins have different AA sequence, the mix of peptides produced is unique to parent protein, these will have unique mass values, so we can find sequence by comparing to database

what is the method for PMF

1. separate individual proteins of interest via gel electrophoresis

2. cut out band of interest containing intact denatured polypeptide chain

3. proteolytically digest with trypsin

4. ionise (protonate) and measure peptide masses using MS

5. compare to database to identify protein

where does trypsin cut?

after K or R amino acids

why is proteomics referred to as post-genimic techonology?

as in order to generate theoretical fingerprint (primary sequence) it is required that the AA sequence being identified is already on database

what is ZooMS?

technique used to identify animal species from ancient or fragmented bone, parchment, or collagen-based artifacts. It works by analyzing collagen peptides through mass spectrometry, comparing their unique fingerprints to reference databases.

what is tandem MS method?

1. 1st analysis to generate peptide fingerprint e.g use PMF

2. collision cell, selects ionised peptide and fragments it futher

3. 2nd analysis, analyses fragmented peptide to generate AA sequence

what does collision cell do in tandem MS?

traps and fragments selected peptide, generates charged ‘daughter ions’ with H+ retained on C-terminal, also produces frayed N-terminus fragments

how is results from collision cell used in tandem MS?

analysed by MS, mass differences between ions can be used to deduce unknown peptide sequences

what is the database and program used in analysis of peptide sequences?

SWISS-PROT database, BLAST programme