Scanning Modes

Question 1

In DIA-SWATH, why are fixed width windows (25m/z) sometimes supobtimal for complex samples?

Answer 1

High mass regions contain fewer peptides, wasting duty cycle

Question 2

In DDA acquisition, why are MS1 scans in a smaller range than MS2?

Answer 2

Because the fragments span a wider range.

Question 3

What is a disadvantage of DDA?

Answer 3

• Poor reproducibility
• ## Low intensity peaks are neglected

Question 4

Why is DIA data harder to analyse?

Answer 4

More data is complex and requires software. Fragments from precursors can overlap.

Question 5

Name the 4 scanning modes that depend on ‘Filtering/Mass Selection’

Answer 5

SIM, Product Ion, Precursor Ion, SRM/MRM

Question 6

Explain SIM in a 3Q

Answer 6

Single Ion Monitoring: Quantitative scane where 1 m/z is monitored in a narrow window. Highly sensitive and selective. Disadvantage: need to know what you are looking for
Q1: Mass selection
Q2: Mass selection
Q3: Mass Selection

Question 7

Explain Product Ion Scan in 3Q

Answer 7

All products of 1 specific precursor ion are measured. Used for Proteomics (DDA): Qualitative& Structural information
Q1: Mass selection
Q2: Collision
Q3: Full scan

Question 8

Explain Precursor Ion Scan in 3Q

Answer 8

Look for precursors that produce a specific diagnostic product ion. For class based detection (e.a. phosphates)
Q1: Full Scan
Q2: Collision
Q3: Mass Selection

Question 9

Explain Neutral Loss Scan in 3Q

Answer 9

Find ions that lose a specific neutral fragment during fragmentation
Q1: Full Scan
Q2: Collision
Q3: Scan across delta m/z lower

Question 10

Explain SRM

Answer 10

Selected Reaction Monitoring, which is targeted detection of a specific precursor & specific fragment ion. Good for sensitive quantitation from complicated mixtures
Q1: Mass Selection
Q2: Collision
Q3: Mass Selection (of multiple?)

Question 11

Why is SRM/MRM the gold standard for quantitation in industry?

Answer 11

• Reproducibility
• Sensitivity
• Specificity
• Straightforward validation

Question 12

Explain the principle of DDA

Answer 12

Data Dependent Acquisition selects the 5 most abundant peaks, and applied product-ion scan to them.

Question 13

Explain why we need Dynamic Exclusion and how it works

Answer 13

In standard DDA, the most abundant peaks are chosen which can lead to missing important information in lower peaks. Therefore: peaks that have been selected are temporarily put on an ‘exclusion list’, preventing it from being fragmented

Question 14

Explain the difference between Gas Phase Fractionation and PAcIFIC to increase sensitivity for DDA

Answer 14

Gas Phase Fractionation: select smaller m/z windows and then inject multiple times. Limitation = time
PACIFIC: Fragment/measure selected precursor ions scan over the whole LC range. Limitation = time

Question 15

Explain the principle of SWATH-MS

Answer 15

Overlapping windows of an m/z range (usually 25 Da) are preselected and MS2 is recorded for each window, per MS1 scan.

Question 16

Explain the advantages of DIA over DDA

Answer 16

• Provides full coverage (of low-abundance and coeluting), while DDA misses this
• Systematic analysis: repeatable
• Unbiased precursor ion selection
• High efficiency
• But complex data! Software is needed

Question 17

Describe the complexity of the proteome

Answer 17

We have many (20k) genes, coding for even more proteins (850k). These proteins also get post-translational modifications, which means at least 10’s of millions different proteins.

Question 18

Why do we prefer DIA for Proteomics?

Answer 18

1. Higher reproducibility
2. Better quantification for complex samples
3. Captures complete quantitative dataset –> helps in discovery proteomics

Question 19

Explain how diaPASEF helped protein discovery

Answer 19

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