Analysis of Reproducibility of Proteome Coverage and Quantitation Using Isobaric Mass Tags (iTRAQ and TMT).
This study aimed to compare the depth and reproducibility of total proteome and differentially expressed protein coverage in technical duplicates and triplicates using iTRAQ 4-plex, iTRAQ 8-plex, and TMT 6-plex reagents. The analysis was undertaken because comprehensive comparisons of isobaric mass tag reproducibility have not been widely reported in the literature. The highest number of proteins was identified with 4-plex, followed by 8-plex and then 6-plex reagents. Quantitative analyses revealed that more differentially expressed proteins were identified with 4-plex reagents than 8-plex reagents and 6-plex reagents. Replicate reproducibility was determined to be ≥69% for technical duplicates and ≥57% for technical triplicates. The results indicate that running an 8-plex or 6-plex experiment instead of a 4-plex experiment resulted in 26 or 39% fewer protein identifications, respectively. When 4-plex spectra were searched with three software tools-ProteinPilot, Mascot, and Proteome Discoverer-the highest number of protein identifications were obtained with Mascot. The analysis of negative controls demonstrated the importance of running experiments as replicates. Overall, this study demonstrates the advantages of using iTRAQ 4-plex reagents over iTRAQ 8-plex and TMT 6-plex reagents, provides estimates of technical duplicate and triplicate reproducibility, and emphasizes the value of running replicate samples.
Proteomics International , Perth, Western Australia 6009, Australia.|Harry Perkins Institute of Medical Research, QEII Medical Centre, The University of Western Australia , Nedlands, Western Australia 6009, Australia.
Tammy M
TM
Casey
Proteomics International , Perth, Western Australia 6009, Australia.|Harry Perkins Institute of Medical Research, QEII Medical Centre, The University of Western Australia , Nedlands, Western Australia 6009, Australia.
Javed M
JM
Khan
Proteomics International , Perth, Western Australia 6009, Australia.
Scott D
SD
Bringans
Proteomics International , Perth, Western Australia 6009, Australia.
Tomas
T
Koudelka
Proteomics International , Perth, Western Australia 6009, Australia.
Pari S
PS
Takle
Proteomics International , Perth, Western Australia 6009, Australia.
Rachael A
RA
Downs
Proteomics International , Perth, Western Australia 6009, Australia.
Andreja
A
Livk
Centre for Crop and Disease Management, Department of Environment and Agriculture, Curtin University , Bentley, Western Australia 6102, Australia.
Robert A
RA
Syme
Centre for Crop and Disease Management, Department of Environment and Agriculture, Curtin University , Bentley, Western Australia 6102, Australia.
Kar-Chun
0000-0001-6094-823X
KC
Tan
Proteomics International , Perth, Western Australia 6009, Australia.
Richard J
RJ
Lipscombe
D001203:Ascomycota / Q000737:chemistry*; D005656:Fungal Proteins / Q000032:analysis* / Q000737:chemistry; D058977:Molecular Sequence Annotation; D010446:Peptide Fragments / Q000032:analysis*; D059748:Proteolysis; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods / Q000592:standards*; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D013194:Staining and Labeling / Q000379:methods; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry
D005656:Fungal Proteins; D010446:Peptide Fragments; D020543:Proteome; D011933:Reagent Kits, Diagnostic; D014357:Trypsin
TMT; iTRAQ; reproducibility