lecture 13 - mass spectrometry

Question 1

why is mass spectrometry useful?

3

Answer 1

(1) measure total mass
(2) measure mass of proteolytic fragments (often identify protein by matching this to a database of known sequences)
(3) sequence short peptides

Question 2

what is mass spectromoetry actually measuring as total mass?

Answer 2

mass/charge ratio (m/z)

Question 3

how does measuring mass by mass spectrometry work?

Answer 3

• charged sample is run through an electric feild
• (charge sample by adding H+ - varries for each molecule)
• detector collects time of arrival of ions to get m/z

Question 4

we can transform relative intensity and m/z to:

Answer 4

• intensity and mass

- can detect differences due to isotopes

Question 5

what are the benefits to mass spectrometry? (3)

Answer 5

(1) very sensetive and accurate measure of mass
(2) can detect post-translational modifications - determines changes in mass compared to protein sequence alone
(3) can count # of disulphide bonds - compare oxidized mass to that of reduced and blocked protein

Question 6

how do we sequence proteolytic peptides?

Answer 6

• using tandem MS (MS/MS)

- 2 linked MS’s

Question 7

summarize how tandem MS works

Answer 7

• start w pure protein
• digest protein with proteases (e.g. trypsin for tryptic polypeptides)
• analyze peptides with MS/MS

Question 8

why is Tandem MS useful?

Answer 8

no need to purify or separate peptides

Question 9

what are the parts of Tandem MS?

Answer 9

• proteolytic mix travel through:
• MS1
• collision cell
• MS2
• to detector

Question 10

what does MS1 do?

Answer 10

selects one peptide (one m/z)

Question 11

what does the collision cell do?

Answer 11

• collisions with the selected peptide result in fragmentation at certain bonds (moslty @ peptide bonds)
• typically one peptide bond is broken per peptide molecule
• this creates a mixture of all possible fragments
• not hydrolysis ** no addition of h2o

Question 12

what does MS2 do?

Answer 12

• analyzes fragments

- gives m/z (masses)

Question 13

example question: R1 R2 R3 R4 peptide undergoes collisions, what is the result?

Answer 13

R1 | R2 R3 R4
R1 R2 | R3 R4
R1 R2 R3 | R4
R1 R2 R3 R4

Question 14

example question: Phe Pro Ser Ala Arg

Arg = 172 Da
Ala Arg = 243 Da
Ser Ala Arg = 330 Da
Pro Ser Ala Arg = 427 Da
Phe Pro Ser Ala Arg = 574 Da 

each individual protein in Da?

Answer 14

Ala = 71 Da
Ser = 87 Da 
Pro = 97
Phe = 147

Note: these differences are the residue mass (amino acid mass -18 Da)
Note: C-term res has OH added to residue mass

Question 15

What amino acids cannot be distinguished by MS/MS?

Why?

Answer 15

• Leu & Ile

- identical masses