Proteomic analysis of prolactinoma cells by immuno-laser capture microdissection combined with online two-dimensional nano-scale liquid chromatography/mass spectrometry

Abstract

Background: Pituitary adenomas, the third most common intracranial tumor, comprise nearly 16.7% of intracranial neoplasm and 25%-44% of pituitary adenomas are prolactinomas. Prolactinoma represents a complex heterogeneous mixture of cells including prolactin (PRL), endothelial cells, fibroblasts, and other stromal cells, making it difficult to dissect the molecular and cellular mechanisms of prolactin cells in pituitary tumorigenesis through high-throughout-omics analysis. Our newly developed immuno-laser capture microdissection (LCM) method would permit rapid and reliable procurement of prolactin cells from this heterogeneous tissue. Thus, prolactin cell specific molecular events involved in pituitary tumorigenesis and cell signaling can be approached by proteomic analysis.

Results: Proteins from immuno-LCM captured prolactin cells were digested; resulting peptides were separated by two dimensional-nanoscale liquid chromatography (2D-nanoLC/MS) and characterized by tandem mass spectrometry. All MS/MS spectrums were analyzed by SEQUEST against the human International Protein Index database and a specific prolactinoma proteome consisting of 2243 proteins was identified. This collection of identified proteins by far represents the largest and the most comprehensive database of proteome for prolactinoma. Category analysis of the proteome revealed a widely unbiased access to various proteins with diverse functional characteristics.

Conclusions: This manuscript described a more comprehensive proteomic profile of prolactinomas compared to other previous published reports. Thanks to the application of immuno-LCM combined with online two-dimensional nano-scale liquid chromatography here permitted identification of more proteins and, to our best knowledge, generated the largest prolactinoma proteome. This enlarged proteome would contribute significantly to further understanding of prolactinoma tumorigenesis which is crucial to the management of prolactinomas.

Figure 1

SDS-PAGE of PRL cells. PRL cells collected from LCM were lyzed with urea (7 M) and thiourea (2 M). Solubilized proteins were separated via SDS-PAGE and stained with Coomassie blue. The gel was cut into five sections based on molecular mass and each section was subjected to in-gel digestion with trypsin.

Figure 2

Categories of identified proteins in prolactinomas by cell components. The distribution of enrolled proteins in DAVID 2009 was 16% of cell part, 15% of intracellular part, 15% of intracellular, 12% of vesicle, 4% of protein complex and other 11 fractions according to cell components.

Figure 3

Protein overlaps of two proteomes. Total proteins are indicated for each proteome with two circles; subsets in common (in blue) are indicated within the diagram. It indicates that most proteins identified in 2-DE (in brown) are overlapped with what extracted from the prolactinomas by immuno-LCM (in green).

Figure 4

Categories of identified proteins in prolactinomas by biological process. Proteins in the proteome participate in pituitary hormones, cellular signals, biosynthetic process, cellular metabolic process, cell development, response to endogenous stimulus, transportation, etc.