b'SureQuant assays and standardsValidated, modular reagents for multiplexed targetedHighlights:protein quantitation Completeincludes reagents for successful sample The Thermo Scientific SureQuant assays and standardspreparation and quantitative analysis of each target peptidehave been designed for multiplexed targeted quantitation forVerifiedkits and reagents rigorously tested for specificity and key cellular signaling proteins. The assays provide flexibility tosuccessful quantitation of each target peptidechoose the use of the immunoprecipitation sample prep kits andMultiplexThermo Scientific HeavyPeptide AQUA peptide quantitation modules together or independently to bestpanels for simultaneous quantitation of target proteins and suit the end application. The MS sample preparation has beenphosphorylation status from key cell signaling pathwaysstreamlined to be completed in ~4 hours. Together, SureQuant assays and standards provide a complete solution for multiplex IPFlexiblemodular format allows for immunoenrichment only, or in combination peptide quantitation panelsenrichment and analysis of target proteins (Figures 4 and 5).Learn more at thermofisher.com/ms-targeted-assaysA B90 90 12 12Large intestineL anrogrem ianltestine normal Breast normalBreast normal80 80 Breast tumor Breast tumorLarge intestineL aturgmeo irntestine tumor 10 1070 7060 60 8 8fmol target fmol target fmol target fmol target50 5040 40 6 630 30 4 420 2010 10 2 20 0 0 0GSS1K3 GSS1K3AKT1 AKT2 AKATK3T1AKTA1KST12GSAKK3T3GSAK3T1S1IGGFS1RK3 IR mTIGOFR1R PTIERNS1 KSm6TBO1R TSPCT2EN KS6B1 TSC2 AKT1 AKT2 AKATK3T1AKTA1KST12GSAKK3T3GASK3T1S1IGGFS1RK3 IR mTIGOFR1R PTIERNS1 KSm6TBO1R TSPCT2EN KS6B1 TSC2Figure 4. Multiplex IP analysis using streptavidin magnetic beads with human tissue. AKT/mTOR pathway proteins were enriched through multiplex IP using biotinylated antibodies from Thermo Scientific SureQuant AKT pathway kits. Parallel reaction monitoring (PRM) analysis for (A) total AKT/mTOR pathway targets and (B) phosphorylated AKT/mTOR pathway targets was performed using the Thermo Scientific UltiMate 3000 RSLCnano System and Thermo Scientific Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer. Data were subsequently analyzed in Skyline software using calibration curves to determine the absolute protein concentration (fmol).8.0 x 106 6.0 x 107 1.6 x 108Row Z-Score AKT1:S473 STAT3:Y705 IRS1:S11017.0 x 106 5.0 x 107 1.4 x 1081 0 +189 6.0 x 106 1.2 x 10831115 4 6 4.0 x 107 8233523 5.0 x 10 1.0 x 10128119914195104103 5 4.0 x 106 3.0 x 107 8.0 x 10783606513381278 3.0 x 106 7 6.0 x 10787110100 2.0 x 103756498681 2.0 x 106 4.0 x 10752855412912730 6 1.0 x 107 712359 1.0 x 10 2.0 x 10847571741011107106 0.0 x 106 0.0 x 107 0.0 x 1074613021 A B C D E A B C D E A B C D E7266333239 928179916 8 7502227111 1.2 x 106 3.5 x 107 2.5 x 1076462256973 STAT1:Y701 FOXO3:S294 TSC2:S939122936788114 6 3.0 x 107125 1.0 x 10 734109 2.0 x 1011896364810543 3 5 2.5 x 1075557131 8.0 x 102019126 7 1.5 x 1079279 2.0 x 10531208210858 6.0 x 10540511576 1.5 x 107 711277 1.0 x 106812418121 4.0 x 1052990101 6 1.0 x 1076394116 145 5 5.0 x 106117102 2.0 x 10 611314 5.0 x 106170424780249897 2 6A B C D E 44 0.0 x 105 A B C D E 0.0 x 106 A B C D E 0.0 x 10 A B C D EFigure 5. Use of the Thermo Scientific SureQuant Multipathway Phosphopeptide Standard with phosphopeptide enrichment enables relative quantitation of greater than 100 phosphoproteins. Heat map of relative levels of phosphopeptides (left). Changes in specific phosphopeptides (right). One microgram of treated cell lines was incubated with Fe-NTA magnetic beads. Prior to enrichment, 1 pmol of the SureQuant Multipathway Phosphopeptide Standard was spiked into the lysate. PRM analysis was performed using the UltiMate 3000 RSLCnano System and Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer and subsequently analyzed in Skyline software using the average total area. A: HCT116/IGF; B: MCF7/IGF; C: LNCaP/IGF; D: A431/EGF; E: HepG2/Ins. 92 Mass spectrometry thermofisher.com/proteinbiology'