Dataset Information

Global Proteomic Analysis of Functional Compartments in Immature Avian Follicles Using Laser Microdissection Coupled to LC-MS/MS.

ABSTRACT: Laser microdissection (LMD) was utilized for the separation of the yolk, follicular wall (granulosa and theca), and surrounding stromal cells of small white follicles (SWF) obtained from reproductively active domestic fowl. Herein, we provide an in situ proteomics-based approach to studying follicular development through the use of LMD and mass spectrometry. This study resulted in a total of 2889 proteins identified from the three specific isolated compartments. White yolk from the smallest avian follicles resulted in the identification of 1984 proteins, while isolated follicular wall and ovarian stroma yielded 2470 and 2456 proteins, respectively. GO annotations highlighted the functional differences between the compartments. Among the three compartments examined, the relative abundance of vitellogenins, steroidogenic enzymes, anti-Mullerian hormone, transcription factors, and proteins involved in retinoic acid receptors/retinoic acid synthesis, transcription factors, and cell surface receptors such as EGFR and their associated signaling pathways reflected known cellular function of the ovary. This study has provided a global proteome for SWF, white yolk, and ovarian stroma of the avian ovary that can be used as a baseline for future studies and verifies that the coupling of LMD with proteomic analysis can be used to evaluate proteins from small, physiologically functional compartments of complex tissue.

SUBMITTER: Nepomuceno AI

PROVIDER: S-EPMC4671083 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Publications

Global Proteomic Analysis of Functional Compartments in Immature Avian Follicles Using Laser Microdissection Coupled to LC-MS/MS.

Nepomuceno Angelito I AI Muddiman David C DC Petitte James N JN

Journal of proteome research 20150812 9

Laser microdissection (LMD) was utilized for the separation of the yolk, follicular wall (granulosa and theca), and surrounding stromal cells of small white follicles (SWF) obtained from reproductively active domestic fowl. Herein, we provide an in situ proteomics-based approach to studying follicular development through the use of LMD and mass spectrometry. This study resulted in a total of 2889 proteins identified from the three specific isolated compartments. White yolk from the smallest avia ...[more]

PMID: 26211554

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Project description:Background:Haematoxylin and eosin (H&E)-which respectively stain nuclei blue and other cellular and stromal material pink-are routinely used for clinical diagnosis based on the identification of morphological features. A richer characterization can be achieved by laser capture microdissection coupled to mass spectrometry (LCM-MS), giving an unbiased assay of the proteins that make up the tissue. However, the process of fixing and H&E staining of tissues provides challenges with standard sample preparation methods for mass spectrometry, resulting in low protein yield. Here we describe a microproteomics technique to analyse H&E-stained, formalin-fixed paraffin-embedded (FFPE) tissues. Methods:Herein, we utilize heat extraction, physical disruption, and in column digestion for the analysis of H&E stained FFPE tissues. Micro-dissected morphologically normal human lung alveoli (0.082 mm3) and human lung blood vessels (0.094 mm3) from FFPE-fixed H&E-stained sections from Idiopathic Pulmonary Fibrosis (IPF) specimens (n?=?3 IPF specimens) were then subject to a qualitative and then quantitative proteomics approach using BayesENproteomics. In addition, we tested the sensitivity of this method by processing and analysing a range of micro-dissected human lung blood vessel tissue volumes. Results:This approach yields 1252 uniquely expressed proteins (at a protein identification threshold of 3 unique peptides) with 892 differentially expressed proteins between these regions. In accord with prior knowledge, our methodology approach confirms that human lung blood vessels are enriched with smoothelin, CNN1, ITGA7, MYH11, TAGLN, and PTGIS; whereas morphologically normal human lung alveoli are enriched with cytokeratin-7, -8, -18, -19, 14, and -17. In addition, we identify a total of 137 extracellular matrix (ECM) proteins and immunohistologically validate that laminin subunit beta-1 localizes to morphologically normal human lung alveoli and tenascin localizes to human lung blood vessels. Lastly, we show that this micro-proteomics technique can be applied to tissue volumes as low as 0.0125 mm3. Conclusion:Herein we show that our multistep sample preparation methodology of LCM-MS can identify distinct, characteristic proteomic compositions of anatomical features within complex fixed and stained tissues.

Dataset Information

Global Proteomic Analysis of Functional Compartments in Immature Avian Follicles Using Laser Microdissection Coupled to LC-MS/MS.

Publications

Global Proteomic Analysis of Functional Compartments in Immature Avian Follicles Using Laser Microdissection Coupled to LC-MS/MS.

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