Can not see Light/Endogeneous peptides | VM26 | 2024-03-20 18:28 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Hi All, I have been trying to develop a PRM assay for 1 protein for which I have 3 Heavy peptides. I have been able to develop a linear curve for LOD and LOQ using CSF (fixed amount) as a matrix with varying conc of Heavy peptides. Using Light as the internal standard and heavy as Isotopically labeled, I could plot a linear graph for 1 peptide. However, I was unable to get a linear curve for 2 peptides of the same protein. However, I am unsure how to proceed with 1:1. CASE 1 CASE 2 In Case 1; my every dilution vial has 5ul CSF (fixed volume so fixed amount of CSF) and 5ul from respective dilution tube (1000.....0.25fmol/ul) In Case 2; my main mixed stock of 5ul CSF+ 5ul 2500fmol/ul is serving as highest stock and I am diluting using this as the main vial and 0.1% Formic acid as diluent. Now, I am unsure which will work best as my endogenous peptides are way low but get overexpressed in the disease model of my interest. CSF used here is the pool of 20 patients to avoid heterogeneity. Can you suggest some solution. Thank you. |
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