MALDI-TOF MS Analysis of Protein Samples

Samples were prepared as described previously [10 (link)] with some modifications. Briefly, a modified Lowry protein assay was used to measure total protein concentrations in the individual and pooled sample in each group [25 ]. Dried pooled samples were reconstituted in acetonitrile (ACN) with 5% (v/v) trifluoroacetic acid (TFA) before mixing with an equal volume of MALDI matrix (10 mg/mL α-cyano-4-hydroxycinnamic acid in 100% ACN containing 5% TFA). Sixteen replicates were spotted on to MALDI target plates (Bruker Daltonics, Billerica, MA, USA). Mass spectra were obtained using an Ultraflex III TOF/TOF instrument (Bruker Daltonics) in a linear positive mode within a mass range of 1000 to 20,000 Da. An external calibration was performed using a Proteo-Mass Peptide and Protein MALDI-MS Calibration Kit (Sigma Aldrich, St. Louis, MO, USA). A total of 500 laser shots at 50 Hz were used to generate each spectrum. Peptide mass spectra were processed using flexAnalysis v. 3.3 software, whereas the PMF and PCA of target mass spectra between 1000 and 20,000 Da were analyzed using ClinProTools v. 3.0 software [26 (link)–28 (link)]. The reliability and the accuracy of the candidate peak selection were evaluated by > 90% recognition capability and cross-validation values [28 (link)].