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Shotgun proteomics reveals biochemistry of normal and acute respiratory distress syndrome bronchoalveolar lavage fluid proteome
Critical Care volume 12, Article number: P292 (2008)
Recently, so-called label-free quantitative proteomic methods have gained acceptance for protein expression analysis via mass spectrometry (MS). These methods allow better experiment design by circumventing the need to establish pair-wise comparisons required by popular stable isotope dilution methods (for example, isotope coded affinity tag, isobaric tags for relative and absolute quantitation and stable-isotope labelling by amino acids in cell culture) and are thus fundamentally better suited for proteome studies where sample number is large. Here we discuss the use of shotgun proteomics (that is, no prior protein fractionation) and label-free quantitative methods to characterize human bronchoalveolar lavage fluid (BALF) proteomes of six normal healthy volunteers and three patients with acute respiratory distress syndrome (ARDS) .
Our proteomic profiling technology for BALF involves: (i) removal of cells by centrifugation followed by precipitation of the remaining soluble protein, (ii) denaturation and proteolysis of soluble proteins, (iii) analysis in quadruplicate of each sample by LC-MS/MS using Fourier transform-mass spectrometry (FT-MS), (iv) identification of proteins by database search of tandem mass spectra (MS2 data), and (v) extraction of changes in protein relative expression directly from peptide ion intensity values (MS1 data) as recently described . Notably, data for each sample are acquired independent of all other samples, allowing any patient's data to be compared directly with all others in silico. We also measured levels of several proteins of interest (identified in LC-MS/MS experiments) by ELISA in ARDS BALF from 69 patients ranging from those at risk to 14 days post diagnosis and six normals. Enriched functional categories within BALF proteins relative to the entire human proteome were determined using Expression Analysis Systematic Explorer (EASE) software .
Using BALF from three patients, we identified a total of 870 different proteins, a nearly 10-fold increase from previous reports. Among the proteins identified were known markers of lung injury, such as surfactant, proteases, and serum proteins. We also identified several biologically interesting proteins not previously identified in patients with ARDS, including insulin-like growth factor-binding protein-3 (IGFBP-3). Immunoassay showed elevated levels of IGFBP-3 and IGF-I in at-risk patients and those with early ARDS, whereas normal controls had low levels of IGFBP-3. The IGF pathway, acting through the type 1 IGF receptor, repressed apoptosis of lung fibroblasts but not lung epithelial cells. Furthermore, depletion of IGF in ARDS BALF led to enhanced fibroblast apoptosis. Additionally, normal human BALF was profiled from six volunteers. From these analyses a total of 167 unique proteins were detected with >100 proteins detected in each of the six individual BAL samples, 42 of which were in common to all six subjects.
Our data suggest that the IGFBP-3/IGF pathway is involved in the pathogenesis of lung injury, illustrating the power of shotgun proteomics to catalog proteins present in complex biological fluids, such as BALF, from which hypotheses can be developed and tested. From the normal BALF proteome data, gene ontology analysis demonstrated enrichment of several biological processes in the lung that reflects its expected role in gas exchange and host defense as an immune organ. The same biological processes were enriched compared with either human plasma proteome or total human genome calculated proteome, suggesting an active enrichment of plasma proteins in the lung rather than passive capillary leak.
Schnapp , et al.: Mining the acute respiratory distress syndrome proteome: identification of the insulin-like growth factor (IGF)/IGF-binding protein-3 pathway in acute lung injury. Am J Pathol 2006, 169: 86-95. 10.2353/ajpath.2006.050612
Foss , et al.: Genetic basis of proteome variation in yeast. Nat Genet 2007, 39: 1369-1375. 10.1038/ng.2007.22
Hosack , et al.: Identifying biological themes within lists of genes with EASE. Genome Biol 2003, 4: R70. [http://david.niaid.nih.gov/david/ease.htm] 10.1186/gb-2003-4-10-r70
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Goodlett, D. Shotgun proteomics reveals biochemistry of normal and acute respiratory distress syndrome bronchoalveolar lavage fluid proteome. Crit Care 12, P292 (2008). https://doi.org/10.1186/cc6513
- Acute Respiratory Distress Syndrome
- Bronchoalveolar Lavage Fluid
- Shotgun Proteomics
- Expression Analysis Systematic Explorer
- Enrich Functional Category