Methods in molecular biology
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Patients with severe traumatic brain injury (TBI) frequently present with concomitant injuries that may cause secondary brain injury and impact outcomes. Animal models have been developed that combine contemporary models of TBI with a secondary neurologic insult such as hypoxia, shock, long bone fracture, and radiation exposure. ⋯ Here, we review these models and their collective contribution to the literature on TBI. In addition, we provide protocols and notes for two well-characterized models of TBI plus hemorrhagic shock.
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Protein kinases are widely considered to be invaluable target enzymes for drug discovery and for diagnosing diseases and assessing their prognosis. Effective analytical techniques for measuring the activities of cellular protein kinases are therefore required for studies in the field of phosphoproteomics. We have recently developed a highly sensitive microarray-based technique for tracing the activities of protein kinases. ⋯ In this chapter, we describe a standard protocol for detecting phosphopeptides by biotin-labeled Phos-tag. We also describe a microarray system for high-throughput profiling of intracellular protein kinase activities. The Phos-tag-based method is expected to be useful in the rapid detection of the complex range of phosphorylation reactions involved in cellular signaling events, and it has potential applications in high-throughput screening of kinase activators or inhibitors.
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Obtaining high phosphoproteome coverage requires specific enrichment of phosphorylated peptides from the often extremely complex peptide mixtures generated by proteolytic digestion of biological samples, as well as extensive chromatographic fractionation prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Due to the sample loss resulting from fractionation, this procedure is mainly performed when large quantities of sample are available. To make large-scale phosphoproteomics applicable to smaller amounts of protein we have recently combined highly specific TiO2-based phosphopeptide enrichment with sequential elution from immobilized metal affinity chromatography (SIMAC) for fractionation of mono- and multi-phosphorylated peptides prior to capillary scale hydrophilic interaction liquid chromatography (HILIC) based fractionation of monophosphorylated peptides. In the following protocol we describe the procedure step by step to allow for comprehensive coverage of the phosphoproteome utilizing only a few hundred micrograms of protein.
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Titanium dioxide (TiO2) has very high affinity for phosphopeptides and in recent years it has become one of the most popular methods for phosphopeptide enrichment from complex biological samples. Peptide loading onto TiO2 resin in a highly acidic environment in the presence of 2,5-dihydroxybenzoic acid (DHB), phthalic acid, lactic acid, or glycolic acid has been shown to improve selectivity significantly by reducing unspecific binding of non-phosphorylated peptides. ⋯ TiO2 chromatography is extremely tolerant towards most buffers used in biological experiments, highly robust and as such it has become the method of choice in large-scale phosphoproteomics. Here we describe a batch mode protocol for phosphopeptide enrichment using TiO2 chromatographic material followed by desalting and concentration of the sample by reversed phase micro-columns prior to downstream MS and LC-MS/MS analysis.
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Reversible protein phosphorylation is a key regulatory posttranslational modification that plays a significant role in major cellular signaling processes. Phosphorylation events can be systematically identified, quantified, and localized on protein sequence using publicly available bioinformatic tools. Here we present the software tools commonly used by the phosphoproteomics community, discuss their underlying principles of operation, and provide a protocol for large-scale phosphoproteome data analysis using the MaxQuant software suite.