Combined Effect of Tissue Stabilization and Protein Extraction Methods on Phosphoprotein Analysis

Kofanova, Olga; Fack, Fred; Niclou, Simone P.; Betsou, Fay

doi:10.1089/bio.2013.0008

Cited by 4 publications

(4 citation statements)

References 12 publications

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“…Aside of NFκB and Erk1, PI3K/Akt/mTor signaling pathways regulate neutrophil function and recruitment ( 35 , 36 ). Therefore, we employed a bead-based Bio-Plex assay (Bio-Rad) to simultaneously measure the phosphorylation status of multiple signaling proteins in the same sample ( 37 ). We examined phosphorylation levels of NFκB (p65 Ser 536 ), ERK1/2 (Thr 202 /Tyr 204 , Thr 185 /Tyr 187 ), Akt (Ser 473 ), and mTOR (Ser 2448 ) in LPS stimulated primary human neutrophils ( Figures 2C–F ).…”

Section: Resultsmentioning

confidence: 99%

TRPM7 Kinase Is Essential for Neutrophil Recruitment and Function via Regulation of Akt/mTOR Signaling

et al. 2021

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During inflammation, neutrophils are one of the first responding cells of innate immunity, contributing to a fast clearance of infection and return to homeostasis. However, excessive neutrophil infiltration accelerates unsolicited disproportionate inflammation for instance in autoimmune diseases such as rheumatoid arthritis. The transient-receptor-potential channel-kinase TRPM7 is an essential regulator of immune system homeostasis. Naïve murine T cells with genetic inactivation of the TRPM7 enzyme, due to a point mutation at the active site, are unable to differentiate into pro-inflammatory T cells, whereas regulatory T cells develop normally. Moreover, TRPM7 is vital for lipopolysaccharides (LPS)-induced activation of murine macrophages. Within this study, we show that the channel-kinase TRPM7 is functionally expressed in neutrophils and has an important impact on neutrophil recruitment during inflammation. We find that human neutrophils cannot transmigrate along a CXCL8 chemokine gradient or produce reactive oxygen species in response to gram-negative bacterial lipopolysaccharide LPS, if TRPM7 channel or kinase activity are blocked. Using a recently identified TRPM7 kinase inhibitor, TG100-115, as well as murine neutrophils with genetic ablation of the kinase activity, we confirm the importance of both TRPM7 channel and kinase function in murine neutrophil transmigration and unravel that TRPM7 kinase affects Akt1/mTOR signaling thereby regulating neutrophil transmigration and effector function. Hence, TRPM7 represents an interesting potential target to treat unwanted excessive neutrophil invasion.

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Section: Resultsmentioning

confidence: 99%

TRPM7 Kinase Is Essential for Neutrophil Recruitment and Function via Regulation of Akt/mTOR Signaling

et al. 2021

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“…It has been shown to be compatible with proteomics, preserving peptides, proteins and posttranslational modifications. [39][40][41] Moreover, tissue treated in this manner has been shown to be suitable for matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry imaging of peptides and proteins. 42,43 Here, we present an improved LESA FAIMS mass spectrometry imaging workflow, which makes use of multiple static FAIMS analyses at each LESA-sampled location, and apply the approach to the imaging of intact proteins in samples of heat preserved and frozen tissue from rat kidneys and testes.…”

Section: Introductionmentioning

confidence: 99%

LESA MS Imaging of Heat-Preserved and Frozen Tissue: Benefits of Multistep Static FAIMS

et al. 2018

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We have previously demonstrated liquid extraction surface analysis (LESA) high field asymmetric waveform ion mobility spectrometry (FAIMS) mass spectrometry imaging of proteins in thin tissue sections of brain and liver. Here, we present an improved approach which makes use of multiple static FAIMS parameters at each sampled location and allows a significant improvement in the number of proteins, lipids and drugs that can be imaged simultaneously. The approach is applied to the mass spectrometry imaging of control and cassette-dosed rat kidneys. Mass spectrometry imaging of kidneys typically requires washing to remove excess hemoglobin; however, that is not necessary with this approach. Multi-step static FAIMS mass spectrometry resulted in a six-to sixteen-fold increase in the number of proteins detected in the absence of FAIMS, in addition to smaller increases over single step static FAIMS (chosen for optimum transmission of total protein ions). The benefits of multi-step static FAIMS mass spectrometry for protein detection are also shown for sections of testes. The numbers of proteins detected following multistep FAIMS increased between two-and threefold over single step FAIMS, and between two-and fourteen-fold over LESA alone. Finally, to date, LESA mass spectrometry of proteins in tissue has been undertaken solely on fresh frozen samples. In this work, we demonstrate that heat-preserved tissues are also suitable for these analyses. Heat preservation of tissue improved the number of proteins detected by LESA MS for both kidney and testes tissue (by between two-and fourfold). For both tissue types, the majority of the proteins additionally detected in the heat-treated samples were subsequently detected in the frozen samples when FAIMS was incorporated. Improvements in the numbers of proteins detected were observed for LESA FAIMS MS for the kidney tissue; for testes tissue fewer total proteins were detected following heat preservation, however approximately one third were unique to the heat preserved samples.

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“…Urea-based buffers are widely used for sample homogenization and has been shown to provide higher protein yields than non-urea buffers 31 . Therefore, we decided to use urea-based buffer and optimize concentration of detergents to facilitate recovery of O -glycans from gastric tissue.…”

Section: Resultsmentioning

confidence: 99%

Sample handling of gastric tissue and O-glycan alterations in paired gastric cancer and non-tumorigenic tissues

Adamczyk

Jin

Połom

et al. 2018

Sci Rep

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Sample collection, handling and storage are the most critical steps for ensuring the highest preservation of specimens. Pre-analytical variability can influence the results as protein signatures alter rapidly after tissue excision or during long-term storage. Hence, we evaluated current state-of-the-art biobank preservation methods from a glycomics perspective and analyzed O-glycan alterations occurring in the gastric cancer tissues. Paired tumor and adjacent normal tissue samples were obtained from six patients undergoing gastric cancer surgery. Collected samples (n = 24) were either snap-frozen or heat stabilized and then homogenized. Glycans were released from extracted glycoproteins and analyzed by LC-MS/MS. In total, the relative abundance of 83 O-glycans and 17 derived structural features were used for comparison. There was no statistically significant difference found in variables between snap frozen and heat-stabilized samples, which indicated the two preservation methods were comparable. The data also showed significant changes between normal and cancerous tissue. In addition to a shift from high sialylation in the cancer area towards blood group ABO in the normal area, we also detected that the LacdiNAc epitope (N,N’-diacetyllactosamine) was significantly decreased in cancer samples. The O-glycan alterations that are presented here may provide predictive power for the detection and prognosis of gastric cancer.

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Combined Effect of Tissue Stabilization and Protein Extraction Methods on Phosphoprotein Analysis

Cited by 4 publications

References 12 publications

TRPM7 Kinase Is Essential for Neutrophil Recruitment and Function via Regulation of Akt/mTOR Signaling

TRPM7 Kinase Is Essential for Neutrophil Recruitment and Function via Regulation of Akt/mTOR Signaling

LESA MS Imaging of Heat-Preserved and Frozen Tissue: Benefits of Multistep Static FAIMS

Sample handling of gastric tissue and O-glycan alterations in paired gastric cancer and non-tumorigenic tissues

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