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Pseudomonas aeruginosa is the predominant cause of mortality in patients with cystic fibrosis (CF). We examined the secretome of an acute, transmissible CF P. aeruginosa (Australian epidemic strain 1-R; AES-1R) compared with laboratory-adapted PAO1. Culture supernatant proteins from rich (LB) and minimal (M9) media were compared using 2-DE and 2DLC-MS/MS, which revealed elevated abundance of PasP protease and absence of AprA protease in AES-1R. CF lung-like artificial sputum medium (ASMDM) contains serum and mucin that generally preclude proteomics of secreted proteins. ASMDM culture supernatants were subjected to 2DLC-MS/MS, which allowed the identification of 57 P. aeruginosa proteins, and qualitative spectral counting was used to estimate relative abundance. AES-1R-specific AES_7139 and PasP were more abundant in AES-1R ASMDM culture supernatants, while AprA could only be identified in PAO1. Relative quantitation was performed using selected reaction monitoring. Significantly elevated levels of PasP, LasB, chitin-binding protein (CbpD), and PA4495 were identified in AES-1R ASMDM supernatants. Quantitative PCR showed elevated pasP in AES-1R during early (18 h) ASMDM growth, while no evidence of aprA expression could be observed. Genomic screening of CF isolates revealed aes_7139 was present in all AES-1 and one pair of sequential nonepidemic isolates. Secreted proteins may be crucial in aiding CF-associated P. aeruginosa to establish infection and for adaptation to the CF lung.

Diagnosis of acute coronary syndromes is based on protein biomarkers, such as the cardiac troponins (cTnI/cTnT) and creatine kinase (CK-MB) that are released into the circulation. Biomarker discovery is focused on identifying very low abundance tissue-derived analytes from within albumin-rich plasma, in which the wide dynamic range of the native protein complement hinders classical proteomic investigations. We employed an ex vivo rabbit model of myocardial ischemia/reperfusion (I/R) injury using Langendorff buffer perfusion. Nonrecirculating perfusate was collected over a temporal profile of 60 min reperfusion following brief, reversible ischemia (15 min; 15I/60R) for comparison with irreversible I/R (60I/60R). Perfusate proteins were separated using two-dimensional gel electrophoresis (2-DE) and identified by mass spectrometry (MS), revealing 26 tissue-specific proteins released during reperfusion post-15I. Proteins released during irreversible I/R (60I/60R) were profiled using gel-based (2-DE and one-dimensional gel electrophoresis coupled to liquid chromatography and tandem mass spectrometry; geLC-MS) and gel-free (LC-MS/MS) methods. A total of 192 tissue-specific proteins were identified during reperfusion post-60I. Identified proteins included those previously associated with I/R (myoglobin, CK-MB, cTnI, and cTnT), in addition to examples currently under investigation in large cohort studies (heart-type fatty acid binding protein; FABPH). The postischemic release profile of a novel cardiac-specific protein, cysteine and glycine-rich protein 3 (Csrp3; cardiac LIM domain protein) was validated by Western blot analysis. We also identified Csrp3 in serum from 6 of 8 patients postreperfusion following acute myocardial infarction. These studies indicate that animal modeling of biomarker release using ex vivo buffer perfused tissue to limit the presence of obfuscating plasma proteins may identify candidates for further study in humans.