2013
DOI: 10.1089/bio.2013.0008
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Combined Effect of Tissue Stabilization and Protein Extraction Methods on Phosphoprotein Analysis

Abstract: Preanalytical conditions applied during sample collection and processing can affect the detection or quantification of unstable phosphoprotein biomarkers. We evaluated the consequences of tissue stabilization and protein extraction methods on phosphoprotein analysis. The effects of stabilization techniques (heat stabilization, snap-freezing) and time on the levels of phosphoproteins, including phospho-Akt, p-ERK 1/2, p-IkBα, p-JNK, and p38 MAPK, were evaluated using a BioPlex phosphoprotein assay. Additionally… Show more

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Cited by 4 publications
(4 citation statements)
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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%
“…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%
“…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%
“…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%