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Figure S1: Comparison of blanks in non-depleted human plasma and depleted human plasma matrices (Supporting Information) 246810121416182022242628303234.

Published byTabitha Shelton Modified over 8 years ago

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Presentation on theme: "Figure S1: Comparison of blanks in non-depleted human plasma and depleted human plasma matrices (Supporting Information) 246810121416182022242628303234."— Presentation transcript:

1 Figure S1: Comparison of blanks in non-depleted human plasma and depleted human plasma matrices (Supporting Information) 246810121416182022242628303234 Time, min 0 50 100 150 200 220 Intensity, cps 246810121416182022242628303234 Time, min 0 5 10 15 20 24 Intensity, cps Non-depleted human plasma tryptic digests Depleted human plasma tryptic digests

2 + CNBr-Activated Sepharose Resin MAb Immobilization of MAb + Ag Antigen Binding Trypsin Scheme 1: Epitope mapping method – Epitope excision followed by LC/MS/MS (Supporting Information) Elution LC/MS/MS Flow Through Lys C if needed Wash

3 Scheme 2: Summary of epitope mapping (Supporting Information) Yellow labels the epitope information provided by Strategic Diagnostics. Red underlines the epitope sequence of TnI-C-2 with its capture MAb. Blue underlines the epitope sequence of TnI-C-2 with its conjugate MAb. MAYATEPHAK KKSKISASRK LQLKTLLLQI AKQELEREAE ERRGEKGRAL STRCQPLELA GLGFAELQDL CRQLHARVDK VDEETSGGGG SGGGGSGGGG SACMDDIYKA AVEQLTEEQK NEFKAAFDIF VLGAEDGCIS TKELGKVMRM LGQNPTPEEL QEMIDEVDED GSGTVDFDEF LVMMVRCMKD DSKGKSEEEL SDLFRMFDKN ADGYIDLDEL KIMLQATGET ITEDDIEELM KDGDKNNDGR IDYDEFLEFM KGVE

4 Scheme 3: Work flow of plasma patient samples (Supporting Information) Results Peptides Mixture Desalting by Waters Oasis 96 well µElution Plate MRM LC/MS/MS, Agilent 1200 capillary HPLC coupled with AB Sciex API 4000 Affinity Purification with Capture MAb coated microparticle Internal Standard Reduced, Alkylated, and Digested Peptides Mixture Plasma Patient Samples Spiked into Normal Human Plasma Digests Matrix

5 Table S1: MRM design Q1 Mass (amu)Q3 Mass (amu)Time (ms)DP (volts)CE (volts) 660.0744.850010037 657.0744.850010037 550.1637.25007526 547.7637.25007526

6 Table S2: Recovery of desalting with 96 well µElution plate (Supporting Information) Targeted Concentration (fmol/µL) DesaltedNon-desaltedRecovery % Analyte (n) Mean of Empirical Concentration (fmol/µL) Analyte (n) Mean of Empirical Concentration (fmol/µL) 12.91912.90213.0199.2 25.81826.44225.93102.0 43.02643.08241.30104.3 64.53963.78265.7597.0 86.04785.44286.0999.2 129.077129.792129.22100.4

7 Table S3: Recovery of normal human plasma matrix (Supporting Information) Targeted Concentration (ng/mL) Solvent Normal human plasma digests Recovery % Analyte (n) Mean of Experimental Concentration (ng/mL) Analyte (n) Mean of Experimental Concentration (ng/mL) 14.92314.96314.0393.8 49.75250.74648.3995.4 99.501100.69398.3397.6 119.402118.918120.91101.7 248.752245.736246.07100.1 497.501499.436497.9799.7

8 Table S4: Plasma patient samples (Supporting Information) Patient Sample Calculated Surrogate Peptide Concentration (ng/mL) Mean Calculated Surrogate Peptide Concentration (ng/mL) % CVMean Calculated Troponin I Concentration (ng/mL) Troponin Concentration by Immunoassay 1 (ng/mL) Troponin Concentration by Immunoassay 2 (ng/mL) A7.91 8.21 7.91 8.01 (n=3) 2.158.3964.8546.58 B7.00 7.63 6.87 7.17 (n=3) 5.752.2646.10QNS* C7.03 7.26 7.12 7.14 (n=3) 2.252.1050.0041.69 * Quantity Not Sufficient

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