Quantification of Isotope Encoded Proteins in 2D Gels

Transcription

1 Quantification of Isotope Encoded Proteins in 2D Gels Using Surface Enhanced Resonance Raman Giselle M. Knudsen 1, Brandon M. Davis 2, Shirshendu K. Deb 1, Yvette Loethen 2, Ravindra Gudihal 1, Pradeep Perera 2, Dor Ben-Amotz 2, V. Jo Davisson 1 * 1 Department of Medicinal Chemistry & Molecular Pharmacology and the Bindley Bioscience Center at Discovery Park and the 2 Department of Chemistry, Purdue University, West Lafayette, IN Supporting Information Available. Supplemental Table 1. MS/MS analysis of peptides matching hgmps species in HCT116 lysate. Supplemental Table 2. MS+MS/MS analysis of peptide coverage of hgmps recombinant protein. Supplemental Table 3. MS/MS identified peptides matching hgmps species in recombinant-spiked sample. Supplemental Figure 1. The fluorescence calibration plot of d 0 -R6G in solution, used for water Raman internal standardization. Supplemental Figure 2. The PLS model calibration plots for absolute versus expected d 0 and d 4 values, d 4 versus d 0 -R6G composition, and %d 4 -R6G composition. Supplemental Figure 3. SERRS spectra from hgmps and background species in Samples A and B, as shown in Figure 6. 1

2 Supplementary Table 1. MS/MS identification of hgmps protein in 2DGE of HCT116 lysate. Putative hgmps protein spots were selected for in-gel tryptic digestion using a protocol from the UCSF mass spectrometry facility ( with zip tip treatment and using α- cyano-4-hydroxycinnamic acid matrix for MALDI preparation. MS/MS analysis was performed using a 4800 MALDI TOF/TOF analyzer from Applied Biosystems, and analyzed using ProteinPilot software v. 2.0, searching the Uniprot+Swissprot databases ( ) with the following settings: trypsin digestion, urea denaturation, iodoacetamide Cys alkylation, species H. sapiens, with special factors: phosphorylation emphasis, gel-based ID, and ID focus biological modifications. The search included proteins, and found only one protein with a score >1.30 (95%): SwissProt# P49915, human GMPS. The number of matched peptides and sequence coverage are listed below. These samples were analyzed through a service provided by the Purdue Proteomics Facility. Sample Unused Total Peptides %Coverage E E E ǂ Protein spot names are labeled in Figure 4B. Although E1 was also analyzed, no significant proteins could be identified from this species. Unused and Total refer to protein scoring used in ProteinPilot software to indicate a summed score for unique peptides that have been unused by other protein hits in the identification, out of the total available. 2

3 Supplementary Table 2. MS+MS/MS analysis of recombinant hgmps species in 2DGE. Samples were selected and digested as described above for MALDI-MS/MS analysis, using an Applied Biosystems MALDI 4700 Protein Analyzer. The 4000 Series Explorer software was used for mass list export into GPS explorer software, searching the NCBInr database ( ) using the Mascot search engine software (v. 2.1) ( Search settings were: combined MS+MS/MS scoring, species H. sapiens, 1 allowed missed cleavage, iodoacetamide modification of Cys, and variable modifications: phospho S,T, Y, Met-ox, Pyro-Glu. The number of matched peptides and sequence coverage are listed for the top hit in all cases: identification of gi , human GMPS. Sample Protein Peptides %C.I. Total Ion Score Score Best Ion Score %C.I. best ion R1 1, , R2 1, , R3 1, ǂ Protein spot names are labeled in Figure 4C. Individual peptide scores in Mascot are significant at >47, the protein score is derived from individual ion scores. 3

4 Supplementary Table 3. MS/MS analysis of Sample A and Sample B protein spots in 2DGE. Protein spots 1-4 were selected for in-gel tryptic digestion, followed by peptide extraction and LC- MS/MS analysis using an Agilent XCT plus coupled with an Agilent 1100 HPLC system. Masses were analyzed using Spectrum Mill software v. A , and searched against the IPI human database, v. 3.00, containing 47,094 sequences. MS/MS analysis settings were: tryptic digest, maximum 2 missed cleavages, fixed carbamidomethyl modification of Cys and variable modifications: phospho Thr/Ser/Tyr, oxidized Met, and pyro-glu. Mass list parameters were precursor mass tolerance 2.5 Da, product mass tolerance 0.7 Da. Shown here are peptide information for identification of P49915, human GMPS. Sample #Spectra Peptides Score %Coverage Mean spectral intensity E E E E+08 ǂ Protein spot names are labeled in Figure 5. 4

5 Supplementary Figure 1. Rhodamine 6G Solution Fluorescence Calibration Plot. Error bars are 1 standard deviation of three replicate measurements for each concentration. The resultant calibration equation is: Normalized Fluorescence Intensity = *(Concentration in pm). Normalized Fluorescence Intensity Concentration (pm) 5

6 Supplementary Figure 2. PLS results obtained using a training set of 1000 Raman spectra with 100%, 75%, 50%, 25%, or 0% d 4 -R6G labeled protein in SDS-PAGE gels stained with silver for surface enhancement of the Raman. The error bars are 95% CI. A linear correlation factor was used to absolutely quantify d 0 -R6G (A) and d 4 -R6G (B). Panel C shows the distribution of d 4 :d 0 compositions, while panel D compares the measured and input (expected) %d 4 composition, with an R 2 correlation coefficient of

7 7

8 Supplementary Figure 3. SERRS spectra collected from protein species in Figure 6 for Samples A and B are shown after baseline correction and Savitsky-Golay smoothing as described in the experimental. The spectra are offset for clarity. Three replicate spectra are shown in each panel for a single protein species, and the panels are arranged in the order: hgmps species 1, 2, 3, 4 background species b1, b2, 8

9 b3, b4 respectively in panels A, B, C, D, E, F, G and H for Sample A and in panels I, J, K, L, M, N, O and P for Sample B. 9