Methods in molecular biology
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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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The impact acceleration (I/A) model of traumatic brain injury (TBI) was developed to reliably induce diffuse traumatic axonal injury in rats in the absence of skull fractures and parenchymal focal lesions. This model replicates a pathophysiology that is commonly observed in humans with diffuse axonal injury (DAI) caused by acceleration-deceleration forces. Such injuries are typical consequences of motor vehicle accidents and falls, which do not necessarily require a direct impact to the closed skull. ⋯ Furthermore, the trauma device is inexpensive and readily manufactured in any laboratory, and the induction of injury is rapid (~45 min per animal from weighing to post-injury recovery) allowing multiple animal experiments per day. In this chapter, we describe in detail the methodology and materials required to produce the rat model of I/A in the laboratory. We also review current adaptations to the model to alter injury severity, discuss frequent complications and technical issues encountered using this model, and provide recommendations to ensure technically sound injury induction.
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Animal models of traumatic brain injury (TBI) provide important tools for studying the pathobiology of brain trauma and for evaluating therapeutic or diagnostic targets. Incorporation of additional insults such as hemorrhagic shock (HS) and/or hypoxemia (HX) into these models more closely recreates clinical scenarios as TBI often occurs in conjunction with these systemic insults (i.e., polytrauma). ⋯ The physiological, histological, and behavioral aspects of this animal model have been characterized and have demonstrated exacerbating effects of systemic insults on penetrating TBI. As such, this model may facilitate the use of simultaneous assessments of multiple mechanisms and provide a platform for testing novel diagnostic and therapeutic targets.
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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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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.