Anlyticl nd Bionlyticl Chemistry Electronic Supplementry Mteril An insight into itraq: where do we stnd now? Croline Evns, Josselin Noirel, Sw Yen Ow, Mlind Slim, An G. Pereir-Medrno, Nrciso Couto, Jgroop Pndhl, Duncn Smith, Trong Kho Phm, Esther Krunkrn, Xin Zou, Ctherine A. Biggs, Phillip C. Wright 1
Tble S1. Assessment of ProteinPilot v3.0 s bckground cpbilities on the 1-to-1, 4 plex bckground dt. Quntifictions published by Ow et l. [7] re compred to those clculted by ProteinPilot v3.0 (PP) for 4- plex itraq protein stndrds. Cytochrome C, bovine serum lbumin, myoglobin nd hen egg lysozyme were mixed t 1:1. Rtios clculted for the Ow et l. [1] row were compred to ProteinPilot (IC nd BC ctivted, column) results nd the theoreticl vlues ( Dilution column). Isotopic correction ws pplied using the isotopic fctors s previously [7]. The closest (best) estimtion of ech column is shded in gry. Protein BSA BSA Dilution Processing b 1 5 5 10 10 100 100 Ow et l. [1] 1.00 4.07 4.17 9.00 8.09 82.2 3 131.85 1.01 4.57 4.79 12.3 13.3 6 0 N/A N/A CYT CYT LYZ LYZ MYO MYO Ow et l. [1] Ow et l. [1] Ow et l. [1] 1.10 4.39 4.20 9.14 8.62 61.0 8 58.40 1.12 5.11 4.74 11.5 13.4 9 3 N/A N/A 0.81 3.79 4.06 8.13 8.36 66.6 6 128.18 0.79 4.25 4.88 11.0 11.0 7 7 N/A N/A 1.05 4.44 4.39 8.28 9.09 68.2 3 104.36 1.05 4.92 4.70 10.8 12.0 6 2 N/A N/A : BSA Bovine Serum Albumin, CYT cytochrome, LYZ Lyzosime, MYO - Myoglobin. b: - ProteinPilot - bckground correction ctivted. N/A = no dt 2
Tble S2 Dilution pttern using four stndrd digested proteins nd liner model used for lestsqures estimtion of protein composition. Smple dilution were mde with either the lbel positions shuffled, or in identicl series using 1:1, 1:5, 1:10, nd 1:100 dilutions. Dgger ( ) shows the highest bundnce lbel. α Dilutions nd lbel positions re shuffled prior to mixing order to ccentute the effects of mixed MS/MS Protein Fctor Lbel 113 Lbel 114 Lbel 115 Lbel 116 Lbel 117 Lbel 118 Lbel 119 Lbel 121 Shuffled Lbel Positions α CYC 100 100 20 20 10 10 1 1 LYZ b 1 1 10 10 20 20 100 100 BSA c 20 10 1 100 100 1 10 20 MYG d 10 20 100 1 1 100 20 10 Resulting pek intensities (theoreticl) 100 + b 1 + c 10 + d 5 100 + b 1 + c 5 + d 10 10 + b 5 + c 1 + d 100 10 + b 5 + c 100 + d 1 5 + b 10 + c 100 + d 1 5 + b 10 + c 1 + d 100 1 + b 100 + c 5 + d 10 1 + b 100 + c 10 + d 5 3
Fig. S1 Anlysis of 121.1 D intensity s function of F residues. A 6 plex itraq nlysis of E. coli lystes, where the 121-D itraq lbel ws not one of the lbels employed. The extrcted intensity (which should in theory red zero) t 121 D directly depends on the presence of phenyllnine in the peptide sequence. The intensity t 121 D for peptides contining either no, one, two nd three phenyllnine residues in the sequence (circled numbers) is plotted. As the number of F residues increses, there is shift in noise detected. Dt were generted using QSTAR XL. 4
Fig. S2 Monitoring MS/MS mixing. A four-protein mix ws t the sme concentrtions s for our 2009 study [7]; however, the dilution pttern for ech protein, cross the vrious lbels, ws kept orthogonl to the other 3 proteins. In this cse, the following dilution ptterns were pplied: 1:1:5:5:10:10:100:100 for lysosyme (s for Ow et l., 2009 [7], 100:100:10:10:5:5:1:1 for cytochrome C, 5:10:100:1:1:100:10:5 for bovine serum lbumin nd 10:5:1:100:100:1:5:10 for myglobin. These ptterns llow for the reltive contribution of ech protein to be determined using lest-squres fitting (Tble S2). Quntifictions were obtined by pseudo-srm nlysis using high resolution ESI TOF/TOF MS (Bruker mxis). A) Peptide contribution to the Cytochrome C peptide itraq+oxidtion* MIFAGI itraq* K elution profile, which reches mximum of 80% t its mximl elution pek. Contmintion from receding elution hed of the pex rises primrily from the recognised lbumin pttern (up to 90%, possibly peptide DAFLGSFLYEYSR chrged 4+) with no more thn 15s between both primry pexes. B) The experimentl rtios s function of time reltive to pex time for the sme cytochrome C peptide: rtios re underestimted throughout the elution of the peptide MIFAGIK but re most ccurte t the pex (blue 1:1 rtio, mgent 1:5 men rtio, gold 1:10 men rtio, green 1:100 men rtio). The elution is shown in grey nd is mesured s the totl intensity of the reporters: the elution of the cytochrome C peptide MIFAGIK only corresponds to the second mode of the pek. 5
Fig S3 Outlier peptides in terms of itraq reltive quntifiction. Cluster nlysis of fold chnge derived from itraq quntifiction in n 8 plex itraq experiment. The blue line indictes the itraq rtios for the lbels 113-121, clculted reltive to 113. For exmple, n upwrd slope indictes up-regultion with respect to 113. The number within prentheses is the number of MS/MS scns for peptide, followed by the peptide sequence. The dt were obtined for the thermosome gmm subunit from Sulfolobus solftricus. It cn be seen tht the mjority of peptides cluster together in terms of reported rtio, with two outlier peptides DIDEIASYLMGK, AEDLLNQK. 6