Lecture 6 - Proteomic analysis

Question 1

What does a trap column do?

Answer 1

Trap non-charged peptides

Question 2

What are the components of Valve B of HPLC?

Answer 2

Injector
Gradient pump
RP trap column
Analytical column
MS
Waste column

Question 3

What does the B ions series retain?

Answer 3

N term

Question 4

What does the Y ion series retain?

Answer 4

C term

Question 5

What does one sequence tag from peptide fragmentation by MSMS include?

Answer 5

4 components:
Peptide molecular weight (MW)
Partial sequence 
Molecular weight before partial sequence
Molecular weight after partial sequence

Question 6

What is MUDPIT?

Answer 6

Multi dimensional protein identification technology

All proteins from an organelle or organism are digested and separated by multidimensional HPLC

Question 7

What does the second valve of MUDPIT do?

Answer 7

Trap charged peptides via. SCX trap column

Question 8

Outline the experimental process of MUDPIT

Answer 8

1) Injection of sample
2) Trap of charged and uncharged peptides via. RP and SCX trap columns
3) Wash away salts
4) RP column is released for data processing and analysis
5) Salt injection
6) Release peptides on SCX column
7) Peptides traped on RP column
8) Wash
9) RP column released for processing and analysis

Question 9

What are key features MUDPIT has to offer?

Answer 9

Separation of very complex mixtures of peptides such as:
Organelles
E.coli lysate
Arabidopsis Chloroplast

Qualitative ID of many proteins

Question 10

Compare the parameters of the following techniques:
MALDI-ToF MS
Liquid chromatography MS

Answer 10

Liquid chromatography has better:
Sensitivity
Dynamic range
Reliability
Coverage
PTM's

MALDI-ToF has better:
Cost efficiency
Time efficiency

Question 11

Outline the difference between MALDI and HPLC

Answer 11

MALDI:
Relies only on the accurate mass of the peptide
Requires many peaks to gain an ID
Works well with coomassie stained protein

HPLC MSMS:
Relies on “nominal” mass of the peptide and its fragmentation
Requires at least 2 peptides matched
Applicable to silver stained proteins routinely

Question 12

What are key features of MALDI ToF ToF?

Answer 12

No separation required
Obtain MSMS data
Very quick and sensitive

But may not get 100% of peptides as they compete for ionisation

Question 13

Why is de novo sequencing (from start) is rarely done?

Answer 13

Relies on bioinformatics to interpret data
Only used if the organism of study is not in protein database
Complicated due to varying charge states and ions other than B and Y series.

Question 14

How can the DeNovo sequencing of peptides be improved?

Answer 14

By using MSn

By doing this, you obtain 2 partial sequence, these sequences can overlap to profile the entire peptide

Question 15

What are the issues of fragmenting peptides?

Answer 15

Other ions are detected such as amino acid side chain fragments or immonium ions (HN=CH-R

These are found on the following peptides:
Proline (P) - 70
Valine (V) - 72
Leucine (L) - 86
Isoleucine (I) - 86
Methionine (M) - 104 
Histidine (H) - 110
Phenylalanine (F) - 120
Tyrosine (Y) - 136
Tryptophan (W) - 159

Question 16

What are Isobaric amino acids?

Answer 16

Amino acids which have the same mass, This can be overcome by using multiple digestion enzymes

e.g. Lysine 128.13 , Glutamine 128.17

Isomers:
Leucine and isoleucine

Question 17

Why is proline cause an issue with fragmentation of peptides?

Answer 17

R side chain cyclise and bonds to N.
This effectively stabilises Y ion
Hence, less cleavage is achieved