Contact Details:

Operator: Andy Christoforou
Contact: ac587@cam.ac.uk
Date: December 2011

Experiment Details:

Experiment Type: Standard LOPIT experimental design on E14TG2a embryonic stem cells
Sample: E14TG2a mouse pluripotent embryonic stem cells cultured under conditions favouring self-renewal (serum+LIF)
Label: iTRAQ 8-plex

Instrument: LTQ Orbitrap Velos
Scan Mode for Identification: MS2-HCD
Scan Mode for Quantitation: MS2-HCD
Scan Setup: Nth order double play, Top 10 HCD
MS1 Scan: FTMS, resolution = 30000, scan range m/z 380 - 1600
MS2 Scan: FTMS, resolution = 7500, scan range m/z 100 - 2000
Precursor Ion Selection Window: 0.5 Da (very stringent)
Collision Energy: 45% CE HCD (two-stepped, 10% width)

Search Parameters:

Search Engine: Mascot
Search Database: UniProt Mouse
Fixed Modifications: iTRAQ 8-plex (N-term), iTRAQ-8plex (K), Methylthio (C)
Variable Modifications: iTRAQ 8-plex (Y), Oxidation (M)
Enzyme: Trypsin
Max. Missed Cleavages: 2
Decoy Type: None (see Percolator parameters below)
Peptide Charge: 5+
Peptide Tolerance: ± 10 ppm
MS/MS Tolerance: ± 0.2 Da
Instrument: ESI-ORBITRAP-HCD

Percolator Parameters:

Percolator Version Used: None. Percolator was misbehaving when this data was processed so Mascot E-values were used to benchmark ID significance rather than PEPs. Any E-values less than 0.05 were accepted for quantitation. The E-value is less stringent than PEP but my general impression is this has made little difference to the overall quality of data. Although no decoy searches were run for Percolating they were performed to check that the FDR was tolerable at the default Mascot p-value of 0.05.

Additional filtering was applied to the data after percolating to refine protein inference within iSPY. Swissprot accessions were given precedence over trEMBL accessions, and isoforms from the same UniProt accession were collapsed together. These assumptions substantially reduce the redundancy of the database, allowing more "unique" peptides to be taken forward for quantitation.

iSPY Parameters:

Analysis Module: ReporterIons.pm
Protein Inference Module: PEDL.pm
Results Output Format: FullTSV.pm
Posterior Error Probability Threshold: 0.05
Maximum Parent Proteins: 1
Precursor Ion Width: 0.06
Precursor Ion Calculation: Trapezoid
Precursor Selection Width: 0.5
Precursor Selection Calculation: Trapezoid
Tag List: 113.107,114.111,115.108,116.111,117.115,118.111,119.115,120.081,121.122
(Note that 120.081 is the phenylalanine immonium ion and not an iTRAQ reporter ion. It was included as a benchmark for identification)
Tag Area Calculation: SUM
Tag Ion Width: 0.03

Post-Processing:

Unquantifiable spectra (E-value for PSM > 0.05, non-unique sequences, very low ion counts, >2 zero value reporter ions ) removed
Spectra filtered based on several criteria (precursor relative signal, position of switch relative to peak apex, reporter ion intensity) to pick a single "peptidotypic" spectrum per peptide
Peptides were merged into proteins by intensity weighted mean
Organelle specific GO annotations were extracted from the UniProt database. Some proteins have annotation relating to multiple organelles.

QuickAnnotation is used to quickly annotate plots with the existing information.
CYT = Cytosol
PLM = Plasma Membrane
ERT = Endoplasmic Reticulum
GOL = Golgi Apparatus
MIT = Mitochondrion
PER = Peroxisome
LYS = Lysosome
END = Endosome
NUC = Nucleus
RIB = Ribosome
PSO = Proteasome
SKE = Cytoskeleton (not normally used for annotation)
ECM = Extracellular Matrix (not normally used for annotation)
VES = Membrane-bound Vesicles (not normally used for annotation)



