In my proteomic analysis, I have tried to a relative quantitation of proteome using 1D Gel LC/MS following by a widely know spectral counting method called as Normalized spectral abundance factor (NSAF). In the sample preparation basically I have a protein mixture from three biological replicated which has been run on a 1D gel in different lanes. Each gel lane was cut into 10 gel fragment and trypsinized. The peptides were then analyzed on a NanoLC couples to LTQ orbitrap. Once the spectra is searched, the list of proteins identified for each replicate was relatively quantified. This kind of label free spectral counting method basically normalizes each protein identification against its length and then against the sum of spectral counts within each sample. My question is that is it a prerequisite to load equal amount of sample in the MS for each gel band within a sample to be able to quantify the data. I have read that NSAFs can normalize different in between samples however I am not sure if its the case within a sample. Please answer if 1. equal volume loading in MS for each gel band 2. If NSAFs can internally normalize spectral counts within a sample.