Use of Composite Protein Database including Search Result Sequences for Mass Spectrometric Analysis of Cell Secretome

Shin, Jihye; Kim, Gamin; Kabir, Mohammad Humayun; Park, Seong Jun; Lee, Seoung Taek; Lee, Cheolju

doi:10.1371/journal.pone.0121692

Cited by 26 publications

(36 citation statements)

References 15 publications

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“…Thus, proteomic profiling of secretomes by shotgun proteomics is an effective method to further the discovery of serological diagnostic markers. Additionally, to increase the identification of true‐positive human secreted proteins using LC‐MS/MS, tandem mass spectral data were searched against a composite database that included experimentally validated FBS contaminant sequences as well as human sequences …”

Section: Discussionmentioning

confidence: 99%

“…Additionally, to increase the identification of true-positive human secreted proteins using LC-MS ⁄ MS, tandem mass spectral data were searched against a composite database that included experimentally validated FBS contaminant sequences as well as human sequences. (11) As shown in Figure 1(a), our own strategy for the selection of marker candidates was designed. Using this strategy, 1410 human secreted proteins from four BC cell lines were identified (Fig.…”

Section: Discussionmentioning

confidence: 99%

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Identification of ganglioside GM2 activator playing a role in cancer cell migration through proteomic analysis of breast cancer secretomes

et al. 2016

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Cancer cell secretomes are considered a potential source for the discovery of cancer markers. In this study, the secretomes of four breast cancer (BC) cell lines (Hs578T, MCF‐7, MDA‐MB‐231, and SK‐BR‐3) were profiled with liquid chromatography–tandem mass spectrometry analysis. A total of 1410 proteins were identified with less than 1% false discovery rate, of which approximately 55% (796 proteins) were predicted to be secreted from cells. To find BC‐specific proteins among the secreted proteins, data of immunohistochemical staining compiled in the Human Protein Atlas were investigated by comparing the data of BC tissues with those of normal tissues. By applying various criteria, including higher expression level in BC tissues, higher predicted potential of secretion, and sufficient number of tandem mass spectra, 12 biomarker candidate proteins including ganglioside GM2 activator (GM2A) were selected for confirmation. Western blot analysis and ELISA for plasma samples of healthy controls and BC patients revealed elevation of GM2A in BC patients, especially those who were estrogen receptor‐negative. Additionally, siRNA‐mediated knockdown of GM2A in BC cells decreased migration in vitro, whereas the overexpression of GM2A led to an increase in cell migration. Although GM2A as a diagnostic and prognostic marker in BC should be carefully verified further, this study has established the potential role of GM2A in BC progression.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Identification of ganglioside GM2 activator playing a role in cancer cell migration through proteomic analysis of breast cancer secretomes

et al. 2016

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“…The values of the mass spectrometer parameters were as follows: spray voltage, 1.9 kV; temperature of the heated capillary, 250 °C; acquisition cycle, one full scan ( m / z 300–2,000) followed by 10 data‐dependent MS/MS scans; isolation width, 2 m / z ; normalized collision energy, 27%; dynamic exclusion duration, 30 s; inclusion list, m / z values of proteotypic peptides for PGRN. The acquired MS/MS spectra were searched against the UniProt mouse (Jan 2015 release) database including horse albumin and NCBI mouse PGRN sequences using SEQUEST in Proteome Discoverer 1.4 (Thermo Fisher, version 1.4.0.288; Shin et al, ). Two trypsin‐missed cleavages were allowed and the peptide mass tolerances for MS/MS and MS were set to ±0.5 and ±15 ppm, respectively.…”

Section: Methodsmentioning

confidence: 99%

Dedifferentiated Schwann cells secrete progranulin that enhances the survival and axon growth of motor neurons

Hyung

Lee

et al. 2018

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Schwann cells (SCs), the primary glia in the peripheral nervous system (PNS), display remarkable plasticity in that fully mature SCs undergo dedifferentiation and convert to repair SCs upon nerve injury. Dedifferentiated SCs provide essential support for PNS regeneration by producing signals that enhance the survival and axon regrowth of damaged neurons, but the identities of neurotrophic factors remain incompletely understood. Here we show that SCs express and secrete progranulin (PGRN), depending on the differentiation status of SCs. PGRN expression and secretion markedly increased as primary SCs underwent dedifferentiation, while PGRN secretion was prevented by administration of cAMP, which induced SC differentiation. We also found that sciatic nerve injury, a physiological trigger of SC dedifferentiation, induced PGRN expression in SCs in vivo. These results suggest that dedifferentiated SCs express and secrete PGRN that functions as a paracrine factor to support the survival and axon growth of neighboring neurons after injury.

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“…The acquired MS/MS spectra from the cell secretome were searched using SEQUEST (TurboSequest version 27, revision 12) against the composite UniProtKB database (released in March 2012), including the human experimentally validated FBS UniProtKB database [ 27 ]. Two trypsin-missed cleavages were allowed and the peptide mass tolerances for MS/MS and MS were set to ± 0.5 and ± 2 Da, respectively.…”

Section: Methodsmentioning

confidence: 99%

Integrative analysis for the discovery of lung cancer serological markers and validation by MRM-MS

et al. 2017

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Non-small-cell lung cancer (NSCLC) constitutes approximately 80% of all diagnosed lung cancers, and diagnostic markers detectable in the plasma/serum of NSCLC patients are greatly needed. In this study, we established a pipeline for the discovery of markers using 9 transcriptome datasets from publicly available databases and profiling of six lung cancer cell secretomes. Thirty-one out of 312 proteins that overlapped between two-fold differentially expressed genes and identified cell secretome proteins were detected in the pooled plasma of lung cancer patients. To quantify the candidates in the serum of NSCLC patients, multiple-reaction-monitoring mass spectrometry (MRM-MS) was performed for five candidate biomarkers. Finally, two potential biomarkers (BCHE and GPx3; AUC = 0.713 and 0.673, respectively) and one two-marker panel generated by logistic regression (BCHE/GPx3; AUC = 0.773) were identified. A validation test was performed by ELISA to evaluate the reproducibility of GPx3 and BCHE expression in an independent set of samples (BCHE and GPx3; AUC = 0.630 and 0.759, respectively, BCHE/GPx3 panel; AUC = 0.788). Collectively, these results demonstrate the feasibility of using our pipeline for marker discovery and our MRM-MS platform for verifying potential biomarkers of human diseases.

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Use of Composite Protein Database including Search Result Sequences for Mass Spectrometric Analysis of Cell Secretome

Cited by 26 publications

References 15 publications

Identification of ganglioside GM2 activator playing a role in cancer cell migration through proteomic analysis of breast cancer secretomes

Identification of ganglioside GM2 activator playing a role in cancer cell migration through proteomic analysis of breast cancer secretomes

Dedifferentiated Schwann cells secrete progranulin that enhances the survival and axon growth of motor neurons

Integrative analysis for the discovery of lung cancer serological markers and validation by MRM-MS

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