To identify differentially expressed proteins associated with cold exposure, non-label quantification was performed (Fig. 5).
The use of both algorithms allowed the identification of a higher number of regulated proteins, because of slight differences in peptide confidence levels and iTRAQ-label quantification values acquired with the two programs.
Stable isotope labeling quantification is generally not affected by retention time shifts.
cDNA synthesis, cRNA double amplification, labelling, quantification, quality control and fragmentation were performed with an automated system (Caliper Life Sciences NV/SA, Teralfene, Belgium), starting with 10 20 ng total RNA from each sample, all as previously described in detail [ 80, 81].
The shotgun approach followed by labeling or label-free quantification has also been widely implemented in biomarker discovery research.
YPED supports many kinds of data including those from multiple MS instruments, different search engines, and labeled or label-free quantification.
Comparing to labeling-based quantification, label-free quantification avoids labeling steps, offers advantages related to the number of samples and the protein detection sensitivity, and has emerged as a high-throughput method in the investigation of scientific questions with clinical relevance over the years [ 30].
Technical reproducibility of label free quantification was proven to be excellent and label free quantification showed a very high homogeneity between patients (Supplementary Fig. 1C).
Label free quantification with SRM and MRM requires prior information of the peptide behavior of the proteins of interest.
The binding partners of the wild type eIF5B and two mutants were identified by label free quantification (LFQ) using the MaxQuant software.
