iTRAQ-Based and Label-Free Proteomics Approaches for Studies of Human Adenovirus Infections
Workflow for comparisons of iTRAQ-label and label-free quantitations. A549 cell lysates were harvested 24 h post infection (p.i.), and proteins were precipitated by TCA, followed by trypsin digestion. For the iTRAQ-label approach, the tryptic peptides were labeled with iTRAQ-8plex reagents. These included biological replicates for noninfected cells (reporter 113 and 114), for HAdV-B3-infected cells (reporter 115 and 116), for HAdV-C5-infected cells (reporter 117 and 118), and for HAdV-B35-infected cells (reporter 119 and 121, not shown here). Peptides were separated by SCX chromatography following RP chromatography and spotted onto MALDI plates. The mass spectrometric analyses were performed on the MALDI-TOF/TOF instrument. Protein quantitation based on iTRAQ data was performed with PP and Sc+ software. For the label-free approach, protein digests were analyzed with LC-MS/MS without prefractionation. The obtained data was quantified by using PL and PF2 and PF3.