Alzheimer’s disease (AD) is the most common neurodegenerative disease, affecting more than 20 million patients worldwide1, 2
. The brains of AD patients are characterized by two pathological hallmarks: senile plaques composed of amyloid-β and neurofibrillary tangles composed of the microtubule-associated protein tau1, 3
. Accumulated evidence suggests that a key step in AD pathogenesis involves tau hyperphosphorylation that leads to the formation of characteristic neurofibrillary tangles4, 5
. Aberrant phosphorylation of several other proteins such as neurofilaments6
and microtubule-associated protein 1B (MAP1B)7
have also been found to associate with AD pathogenesis. These substrates may be modified by a number of kinases, such as GSK-3β8
, cyclin-dependent kinase 5 (CDK5)8
and microtubule-affinity regulating kinase (MARK)8
. Tau may be modified by multiple kinases in a stereotyped sequence: MARK initiating the early phosphorylation events followed by the action of GSK-3β and CDK5 to modify other sites8
. Also implicated are many other kinases including protein kinase C (PKC), cAMP-dependent protein kinase (PKA), calmodulin-dependent protein kinase (CaMKII), and mitogen-activated protein kinase ERK5, 9, 10
. Conversely, those kinases are capable of phosphorylating numerous substrates other than tau in the AD brain11
. Because of technical challenges in the analysis of the phosphoproteome, it is likely that many phosphorylation events in the development of Alzheimer’s disease have not been identified thus far.
The advances in proteomics technologies, especially in phosphopeptide enrichment strategies and liquid chromatography-tandem mass spectrometry (LC-MS/MS)12, 13
, provide opportunities for systematic investigation of protein phosphorylation. Available strategies include immobilized metal-affinity chromatography (IMAC) enrichment incorporating metal oxides such as Fe3+
, TiO216, 17
, cation and anion exchange chromatography19–21
, antibody capture22–24
, chemical derivation25, 26
, and the combination of these approaches27, 28
. More recently, calcium phosphate precipitation (CPP) has been introduced as a simple alternative method to enrich phosphopeptides, in which the phosphopeptides are pulled down by the formation of an insoluble calcium phosphate deposit29
. By coupling phosphate precipitation with the IMAC procedure, 227 non-redundant phosphorylation sites were identified in a rice sample29
. Although the CPP method alone is column-free and straightforward, it is not clear whether this method without subsequent IMAC steps is sufficient to isolate phosphopeptides for direct LC-MS/MS analysis from complex protein mixtures.
Here we describe the phosphoproteome analysis of postmortem AD brain tissue using CPP enrichment directly coupled with the LC-MS/MS approach. Although the level of protein phosphorylation may be reduced by the activities of phosphatases during extended postmortem intervals30
, we show that in an AD brain with a postmortem interval of as long as 20 hours, significant levels of phosphorylated proteins were still detected utilizing this simple procedure. A total of 551 phosphopeptides (466 phosphorylation sites) were identified in the brain tissue, including 379 on serine and 87 on threonine residues. To our knowledge, this is the largest scale phosphoproteome analysis conducted on human AD brain tissue to date.