Proteome Annotations and Identifications of the Human Pulmonary Fibroblast

Abstract

We hereby report on a three year project initiative undertaken by our research team encompassing large-scale protein expression profiling and annotations of human primary lung fibroblast cells. An overview is given of proteomic studies of the fibroblast target cell involved in several diseases such as asthma, idiopatic pulmonary disease, and COPD. It has been the objective within our research team to map and identify the protein expressions occurring in both activated-, as well as resting cell states. The JGGL database www.2DDB.org has been built around these data, allowing advanced hypothesis building using the interactive query bioinformatic tools developed. Gene ontology has been applied to these annotations, classifying and correlating protein expressions to function. The localization as well as the biological processes involved for the annotations are being presented including an annotation-, and sequence-identification strategy, resulting in close to 2000 protein identities. Both gel based, high resolution 2D-gels, and liquid-phase separation (three-dimensional HPLC), as well as the combination of gel- and LC-based approaches (1D-gels and nano-capillary LC, reversed-phase) were utilized. Protein sequencing and structure identities were acquired by a combination of MALDI-, and electrospray-mass spectrometry techniques. Phenotypical and morphological characterizations were also made for this human disease target cell in both stimulated- and resting-cell states. The use of functional assays that demonstrate the key regulating role of growth factors and cytokine stimuli such as PDGF, TGF-beta, and EGF and the effect of ECM molecules such as Biglycan, are also presented and discussed.

Proteomics of bronchoalveolar lavage fluid

“…Since 10 years, major investigation have been realised on isolated lung cells like macrophages [26], lung fibroblast cells [27], isolated ciliary axonemes [28], cell culture of mesenchymal type [29], transformed human bronchial epithelial cell line [30] and cancer cells [24]. Different comprehensive 2-DE gel databases have been started: recently, the group of Malmstrom et al [31] has undertaken an encompassing large-scale protein expression profiling and annotations of human primary lung fibroblast cells in both activated and resting cells. In the same context, Ostrowski et al [28] have initiated an extensive proteomic analysis of isolated ciliary axonemes by 2-DE gel electrophoresis coupled to LC-MS-MS technology.…”

“…In the same context, Ostrowski et al [28] have initiated an extensive proteomic analysis of isolated ciliary axonemes by 2-DE gel electrophoresis coupled to LC-MS-MS technology. Interesting informations have been obtained on the modifications of the lung cell proteome after different stimuli, such as acute exposure to ozone [26] and Jet fuel exposition [32], transforming growth factor beta addition [27], endothelin-1 stimulation [31] and arsenic transformation [33]. The set of these studies have led to significant advances in the molecular mechanisms of different lung pathologies like asthma, fibrosis and lung cancer.…”

Personalized Medicine and Proteomics: Lessons from Non-Small Cell Lung Cancer

“…61 Studies of protein expression in a variety of biological compartments ranging from sub-cellular to whole organismshavebeenundertakenwiththeseanalyticapproaches. [62][63][64][65][66][67][68][69][70] Some key findings from the HUPO initiatives that impact on methodology include:…”

Autonomous protein sample processing on‐chip using solid‐phase microextraction, capillary force pumping, and microdispensing

“…Proteomic studies by utilizing primary disease cells, have been presented by our group previsously [17,18]. Limitations in absolute annotation from 2-D gels have been a major problem in prior investigations, using MALDI-TOF and peptide mass fingerprinting.…”