OC4 - sequencing and identifying proteins

Question 1

how are proteins sequenced and identified?

Answer 1

sample preparation
separation
ionisation
mass spectrometry
infromatics

Question 2

sample preparation

Answer 2

make copies of cells and extract proteins

Question 3

separation

Answer 3

electrophoresis to separate/organise the proteins
using 2D gel electrophoresis

Question 4

2D gel electrophoresis

Answer 4

separation on a gel in two dimensions
firstly by pH where proteins move to where they are electrically neutral at their isoelectric point.
secondly by size using SDS-PAGE, a detergent which gives the proteins a negative charge; so they all migrate towards a positive charge through the gel, the smaller the protein the faster it travels.

Question 5

isoelectric point

Answer 5

the pH at which the amino acid is electrically neutral

Question 6

SDS-PAGE

Answer 6

sodium dodecyl sulphate polyacrylamide gel electrophoresis

Question 7

ionisation

Answer 7

protein molecules are cleaved by addition of proteases, which cleave the protein into peptides
e.g. trypsin > cleaves at the carboxyl side of lysine and arginine except when followed by proline

Question 8

mass spectrometry

Answer 8

peptides enter mss spectrometer and are broken into fragments, which can be measured by a detector and the sequence of amino acids can now be determined
each peak of a mass spec. graph represents the weight of individual amino acids and is used to determine the sequence of amino acids
e.g. MALDI-TOF

Question 9

MALDI-TOF

Answer 9

Matrix assisted laser desorption ionisation - time of flight
a fragmented peptide sample is loaded onto a matrix and ionised through the use of a high energy laser. the fragmented ions are then separated by mass as they all have equal charge.