Improved Mass Spectrometric Proteomic Profiling of the Secretome of Rat Vascular Endothelial Cells

Pellitteri-Hahn, Molly C.; Miriam, Warren; Didier, Daniela N.; Winkler, Erna; Mirza, Shama P.; Greene, Andrew S.; Olivier, Michael

doi:10.1021/pr060287k

Cited by 54 publications

(63 citation statements)

References 9 publications

(14 reference statements)

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“…Once again, the results presented here represent an approximate 10-fold enhancement over the 16 mouse and 31 human extracellular proteins identified in previously related datasets (9 -11). These results also mark a significant improvement over other attempts to characterize cellular secretomes (12)(13)(14)(15)(16)(17).…”

Section: Enhanced Protein Profiling and Hesc Growth Factorsmentioning

confidence: 59%

“…Several studies of extracellular proteomes (secretomes) (12)(13)(14)(15)(16)(17) identified a small number of extracellular proteins but failed to identify growth factors that were known to be present. One of the main problems inherent in these and other large scale MS-based proteomic studies was that a "singlepass" analysis strategy was employed.…”

Section: Human Embryonic Stem Cells (Hescs)mentioning

confidence: 99%

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An Enhanced Mass Spectrometry Approach Reveals Human Embryonic Stem Cell Growth Factors in Culture

Bendall

Hughes

Campbell

et al. 2009

Molecular & Cellular Proteomics

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The derivation and long-term maintenance of human embryonic stem cells (hESCs) has been established in culture formats that are both dependent and independent of support (feeder) cells. However, the factors responsible for preserving the viability of hESCs in a nascent state remain unknown. We describe a mass spectrometrybased method for probing the secretome of the hESC culture microenvironment to identify potential regulating protein factors that are in low abundance. Individual samples were analyzed several times, using successive mass (m/z) and retention time-directed exclusion, without sampling the same peptide ion twice. This iterative exclusion -mass spectrometry (IE-MS) approach more than doubled protein and peptide metrics in comparison to a simple repeat analysis method on the same instrument, even after extensive sample pre-fractionation. Human embryonic stem cells (hESCs)1 are non-transformed cell lines that can proliferate indefinitely in culture, although maintaining the potential to form all primary human cell types (pluripotency) (1, 2). These cells, which originate from the inner cell mass of pre-implantation blastocysts, represent a unique source of human cells for cell replacement therapies and for creating model human systems for understanding disease and development (3). Like other mammalian ESCs, hESCs were originally derived and propagated on replication-deficient mouse embryonic fibroblast (MEF) feeder cells in serum (2, 4), with varying efficiencies (5). At the heart of this variability is a lack of understanding of the regulatory pathways and growth factors that govern hESC self-renewal and pluripotency (6). This ambiguity restricts the application of hESCs in both research and therapeutic applications.We hypothesize that under optimal hESC culture conditions, there exist autocrine and paracrine growth factors, produced both by the feeder cells and the hESCs themselves, that establish the complex microenvironment required to retain hESC potential in culture. Previous genomic-based studies suggested the presence of such networks of hESC transcriptional regulation (7); however, these networks were not correlated to the extracellular microenvironment that ultimately controls hESC fate. Moreover, prior attempts to identify proteins within the hESC microenvironment using MSbased approaches produced few potential candidate regulators and provided little new insight or tangible improvements upon hESC line derivation and culture (6, 8 -11).From the ‡Don Rix Protein Identification Facility,

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Section: Enhanced Protein Profiling and Hesc Growth Factorsmentioning

confidence: 59%

Section: Human Embryonic Stem Cells (Hescs)mentioning

confidence: 99%

An Enhanced Mass Spectrometry Approach Reveals Human Embryonic Stem Cell Growth Factors in Culture

Bendall

Hughes

Campbell

et al. 2009

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

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“…A number of studies have analyzed the secretomes of normal cell types including endothelial, 5 myeloid, 6 adipocytes, 7 microglia, 8 retinal pigment epithelial cells 9 and MDCK cells. 10 The cancer secretome has also been studied in different cancers including pancreatic, 11 nasopharyngeal, 12 thyroid, 13 lung adenocarcinoma, 14 oral, 15 ovarian, 16 colorectal, 17 breast 18 and melanoma.…”

Section: Resultsmentioning

confidence: 99%

SILAC-based quantitative proteomic approach to identify potential biomarkers from the esophageal squamous cell carcinoma secretome

Kashyap

Harsha

Renuse

et al. 2010

Cancer Biology & Therapy

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“…To observe changes in the secreted protein profiles of melanoma samples following PAEP-silencing, mass spectral analysis was conducted on a Thermo Fisher Orbitrap mass spectrometer (Thermo Fisher, Waltham, MA, USA) with an Agilent 1200 Series Nanoflow LC (Agilent Technologies, Santa Clara, CA, USA) for sample introduction, as previously described (13)(14)(15). Prior to analysis, secreted proteins isolated from PAEP-knockdown and non-targeting knockdown 624.38-Mel cells were digested with trypsin in the presence of a reducing agent.…”

Section: Western Blot Analysismentioning

confidence: 99%

Optimization and establishment of RNA interference-mediated knockdown of the progestagen-associated endometrial protein gene in human metastatic melanoma cell lines

Chai¹,

Qiao²,

Wang³

et al. 2013

Molecular Medicine Reports

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Abstract. Progestagen-associated endometrial protein (PAEP), also termed glycodelin, is a 28-kDa glycoprotein of the lipocalin superfamily that is expressed in a variety of tumors, including gynecological tumors, lung cancer and melanoma. To determine the biological functions of the PAEP gene in tumor development and progression, the production of transient and stable PAEP knockdown cell models is required. In the present study, RNA interference technology was used to silence PAEP gene expression in melanoma and transfection was screened for and the conditions were optimized using fluorescence microscopy, flow cytometry, qPCR and western blot analysis. The results of the present study showed that the transient transfection of melanoma cells with 100 nmol/l PAEP siRNA or lentiviral PAEP small hairpin RNA (shRNA) [multiplicity of infection (MOI), 100 pfu/cell] efficiently knocked down target gene expression. To establish stable PAEP knockdown cell lines, melanoma cells were infected with a low MOI (10 pfu/cell) of lentiviral particles expressing PAEP shRNA. Following puromycin screening, the PAEP gene knockdown efficiency was demonstrated to be >80% in 624-Mel and 624.38-Mel cell lines, which was validated by western blot analysis. Therefore, melanoma cell lines with stable knockdown of PAEP were successfully established and may be used as effective cell models to study the biological functions of the PAEP gene in melanoma.

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Improved Mass Spectrometric Proteomic Profiling of the Secretome of Rat Vascular Endothelial Cells

Cited by 54 publications

References 9 publications

An Enhanced Mass Spectrometry Approach Reveals Human Embryonic Stem Cell Growth Factors in Culture

An Enhanced Mass Spectrometry Approach Reveals Human Embryonic Stem Cell Growth Factors in Culture

SILAC-based quantitative proteomic approach to identify potential biomarkers from the esophageal squamous cell carcinoma secretome

Optimization and establishment of RNA interference-mediated knockdown of the progestagen-associated endometrial protein gene in human metastatic melanoma cell lines

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