tutorial: srm assay generation and data analysis in...

Tutorial: SRM assay generation and

data analysis in Skyline EuPA Bioinformatics Course

November 2013 Olga Schubert

Experimental setup of case study •  Mycobacterium tuberculosis (Mtb) was grown in

shake flask cultures and exposed to hypoxic stress for 0h, 6h, and 48h.

•  We will analyse the relative changes of 6 proteins represented by 1 to 3 peptides each.

•  To improve the identification confidence and quantification accuracy, we spiked for all target peptides a synthetic heavy-labelled reference peptide into each sample.

Tryptic Digest

Protein Extraction

SRM TARGETED data

acquisition

precursor selection

fragmentation fragment selection

0.7 Da

Q1 Q3

0.7 Da

Q2

inte

nsity

time

SWATH-MS data-independent

acquisition

precursor selection

fragmentation scanning

25 Da

inte

nsity

time

Q1 TOF

time time

TARGETED data extraction

Q2

10 ppm LC

LC Retention Time

Inte

nsity

Selected reaction monitoring (SRM)

heavy reference peptides

Q1 Q3 RT Rel Inttrans_ID

693.88 906.52 22.9 100YGFIEGHVVIPR.2.y8_1

777.47 22.9 91

368.16 22.9 77

484.33 22.9 53

385.26 22.9 42

YGFIEGHVVIPR.2.y7_1

YGFIEGHVVIPR.2.b3_1

YGFIEGHVVIPR.2.y4_1

YGFIEGHVVIPR.2.y3_1 693.88

693.88

693.88

693.88

SRM assays for proteins of interest

SRM workflow Step Input Output

SRM assay generation List with proteins or peptides of interest, spectral library

SRM instrument method containing optimal transitions for each protein or peptide

SRM measurement Sample, SRM instrument method

SRM traces (.raw)

SRM data analysis I (peak picking)

SRM traces (.raw) Correctly picked peaks, intensity value for each peptide precursor

SRM data analysis II (statistics)

Intensity value for each peptide precursor

Ratio changes of proteins, standard deviation, p-value, etc.

Tryptic Digest

Protein Extraction

SRM TARGETED data

acquisition

precursor selection


0.7 Da

Q1 Q3

0.7 Da

Q2

inte

nsity

time


acquisition

precursor selection


25 Da

inte

nsity

time

Q1 TOF

time time


Q2

10 ppm LC

LC Retention Time

Inte

nsity




693.88 906.52 22.9 100YGFIEGHVVIPR.2.y8_1

777.47 22.9 91

368.16 22.9 77

484.33 22.9 53

385.26 22.9 42

YGFIEGHVVIPR.2.y7_1

YGFIEGHVVIPR.2.b3_1

YGFIEGHVVIPR.2.y4_1


693.88

693.88

693.88


Skyline user interface SRM transi+ons SRM traces SRM peak area overview SRM reten+on +me overview

Skyline transition and peptide settings

Peak picking and refinement

Once we have loaded the data into Skyline, we usually go at least 3 times thoroughly through our Skyline document: 1.  Peak picking

•  Make sure the correct peak is picked à Check the 6 criteria! •  Make sure the peak boundaries are the same width over all samples. •  Make sure the peak boundaries of precursors belonging to the same

peptide are identical.

2.  Peak refinement •  Are all transitions of good quality and reproducible over all runs and

between heavy and light? If not, remove!

3.  Final check

$me (min)

intensity

5. Correla+on reten+on +me (library or predicted SSRCalc*)

3. Signal intensity

$me (min)

intensity

1. Co-‐elu+on

$me (min)

intensity

2. Peak shape

$me (min)

intensity

6. Correla+on with heavy-‐labeled standard

light heavy

light heavy

$me (min) intensity

4. Correla+on peak intensi+es to library spectra

(dot product)

y6

y9

y7

y11 m/z

intensity

SRM peak identification and quantification

Peak picking and refinement Make sure the correct peak is picked. à Peak should be close to the predicted retention time (if LC ran stably and in the calculator accurate iRTs are available for your target peptides) à The Retention Times and Peak Areas replicate views help to find outliers quickly.


Label-free: If your peak is absent, but you know from other samples where it would be, you can pick the noise in that region.


The same peak width should be defined over all samples. à Click onto the x-axis labels below the peak boundary and drag it to where it should be.


For quantification, interfered transitions should be removed from the peak group. In case of label-based SRM: If you remove transitions from the light you also need to remove them from the heavy precursor and vice versa!

Peak picking and refinement For quantification, interfered transitions should be removed from the peak group. à  Change Peak Areas Replicate View to “Normalized To: Total” à  Label-based: Make sure that you remove the transitions always for heavy and

light, even if they are only interfered in one of them.

Peak picking and refinement For quantification, interfered transitions should be removed from the peak group. à  Change Peak Areas Replicate View to “Normalized To: Heavy” à  Ratio indicated next to each transition should be similar (always the ratio for the

activated chromatogram window is shown)

SRM workflow Step Input Output

SRM assay generation List with proteins or peptides of interest, spectral library

SRM instrument method containing optimal transitions for each protein or peptide

SRM measurement Sample, SRM instrument method

SRM traces (.raw)

SRM data analysis I (peak picking)

SRM traces (.raw) Correctly picked peaks, intensity value for each peptide precursor

SRM data analysis II (statistics)

Intensity value for each peptide precursor

Ratio changes of proteins, standard deviation, p-value, etc.

Tryptic Digest

Protein Extraction

SRM TARGETED data

acquisition

precursor selection


0.7 Da

Q1 Q3

0.7 Da

Q2

inte

nsity

time


acquisition

precursor selection


25 Da

inte

nsity

time

Q1 TOF

time time


Q2

10 ppm LC

LC Retention Time

Inte

nsity




693.88 906.52 22.9 100YGFIEGHVVIPR.2.y8_1

777.47 22.9 91

368.16 22.9 77

484.33 22.9 53

385.26 22.9 42

YGFIEGHVVIPR.2.y7_1

YGFIEGHVVIPR.2.b3_1

YGFIEGHVVIPR.2.y4_1


693.88

693.88

693.88


NH2 COOH F H F I S E L E K

light / endogenous NH2 COOH F H F I S E L E K

13C 15N

9.7 9.7

∆m = 8.0142 Da

heavy / isotopically-labeled peptide

Light and heavy peptide forms exhibit the same physico-chemical properties: -  Retention time and peak shape -  Ionisation efficiency -  Fragmentation / relative transition intensities

Label-based quantification by SRM

ratio light/heavy = 1.37

Control Disease


à Protein is not regulated between control and disease





Control Disease

à Protein is 1.46-fold upregulated in the disease state

Ratio disease vs. control (ratio of ratios): 2.00/1.37= 1.46

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