Lecture 4 - Biological MS

Question 1

What are the detection methods used in HPLC/MS?

Answer 1

Full scan (FS)
Single ion monitoring (SIM)
#Single reaction monitoring (SRM)
*#Constant neutral loss (CNL)
*#Precursor Ion discovery (PID)
#Data dependant acquisition (DDA)

*Only possible in space separated MSMS instruments
# Use tandem MS

Question 2

What is full scan MS?

Advantages/Disadvantages

Answer 2

Detection of EVERY ion - Total ion chromatogram
Searches for known ions in the sample
Does not require mass of the compound (analyte)

Advantages:
No data is “lost”
Mass can be extracted after analysis

Disadvantages:
Less sensitive - unimportant m/z values

Question 3

What is Single ion monitoring (SIM)?

Answer 3

All ions enter the MS, but only the ion of interest is passed to the detector. Similar to full scan MS, but does not waste time looking at other ions. Increased sensitivity

Question 4

What is Single Reaction monitoring (SRM)?

Advantages/Disadvantages

Answer 4

Ions are selected by using 3 adjacent quadrupoles.
This effectively selects for the “characteristics” of the fragments of interest from the analyte.

Hence, this is a similar method to SIM, but further more selective.

Advantages:
Increases selectivity and sensitivity by reducing background noise

Disadvantages:
Only compound of interest can be monitored

Question 5

What is constant neutral loss (CNL)?

Advantages/Disadvantages

Answer 5

Ions are selected by using 3 adjacent quadrupoles, same as SRM. But further increases selectivity by monitoring the expected mass loss between Q1 and Q3. If the expected loss of mass is correct, the data is kept, otherwise the data is discarded.

Q1 and Q3 are scanned together with a set mass difference
Response is obtained if the ions “obeys” the set mass difference.

e.g.
Adenosine Nucleoside > Loss of ribose > Adenosine nucleobase

Advantages:
Increased specificity of analysis
Can look for unexpected compounds in given class

Disadvantages:
Less sensitive than SRM as scans all ions

Question 6

What is precursor ion discovery (PID)?

Answer 6

Ions are selected using 3 adjacent quadrupoles, same as SRM/CNL. But its increased selectivity depends on the expected ion from Q3. If the expected ion is produced from Q3, data is kept, otherwise it is discarded.

Question 7

What are the similarities between CNL and PID analysis?

Answer 7

Both use tandem MS, but dont look for a specific compound like SRM

They recognise characteristic fragmentation that identify specific types of compound.

Both balances the specificity and sensitivity of SRM with theanalysis of more than one compound and even unknown modifications of known species.

Question 8

What is data dependent acquisition (DDA)?

Answer 8

The technique which considers everything MAY be important, all data is kept and generates a lot of data. Commonly used for peptide analysis

Question 9

In terms of QUANTITATION, What is Full scan MS used for?

Answer 9

Unknown analyes

Question 10

In terms of QUANTITATION, What is SIM/SRM used for?

Answer 10

Known analytes

Question 11

In terms of QUANTITATION, What is CNL and PID used for?

Answer 11

Known AND unknown analytes

Question 12

In terms of QUANTITATION, What is DDA used for?

Answer 12

May waste too much time

Question 13

In terms of QUALITATIVE analysis, What is SIM/SRM good for?

Answer 13

too specific

Question 14

In terms of QUALITATIVE analysis, What is CNL/PID good for?

Answer 14

Good for all classes of cmps

Question 15

In terms of QUALITATIVE analysis, What is FS/DDA good for?

Answer 15

Will study everything, DDA in more depth but may sacrifice molecule coverage

Question 16

Rank the following in terms of qualitative and quantitative analysis:

SRM
SIM
CNL/PID
FS
DDA

Answer 16

Best for "I dont know what it is" (Qualitative)
1 - DDA
2 - FS
3 - CNL/PID
4 - SIM
5 - SRM

Best for QUANTITATION:
1 - SRM
2 - SIM
3 - CNL/PID
4 - FS
5 - DDA