Optimized proteomic analysis of a mouse model of cerebellar dysfunction using amine-specific isobaric tags

Abstract

Recent proteomic applications have demonstrated their potential for revealing the molecular mechanisms underlying neurodegeneration. The present study quantifies cerebellar protein changes in mice that are deficient in plasma membrane calcium ATPase 2 (PMCA2), an essential neuronal pump that extrudes calcium from cells and is abundantly expressed in Purkinje neurons. PMCA2-null mice display motor dyscoordination and unsteady gait deficits observed in neurological diseases such as multiple sclerosis and ataxia. We optimized an amine-specific isobaric tags (iTRAQ ™ )-based shotgun proteomics workflow for this study. This workflow took consideration of analytical variance as a function of ion signal intensity and employed biological repeats to aid noise reduction. Even with stringent protein identification criteria, we could reliably quantify nearly 1000 proteins, including many neuronal proteins that are important for synaptic function. We identified 21 proteins that were differentially expressed in PMCA2-null mice. These proteins are involved in calcium homeostasis, cell structure and chromosome organization. Our findings shed light on the molecular changes that underlie the neurological deficits observed in PMCA2-null mice. The optimized workflow presented here will be valuable for others who plan to implement the iTRAQ method.

Addressing Accuracy and Precision Issues in iTRAQ Quantitation

“…In particular, the coefficient of variation (CV) is higher in data from low intensity peaks than in data from high intensity peaks (16,22,23). This has also been observed in other MS-based quantitation techniques when quantifying from the MS signal (28 -30).…”

“…Filtering, outlier removal, and weighted methods are of limited use for peptides for which only a few low intensity readings were made; however, such cases typically dominate the data sets. Even with a logarithmic transformation, heterogeneity has been reported for iTRAQ data (16,19,22). Current methods struggle to address the issue and to maintain sensitivity.…”

“…Some of the current approaches include outlier removal (18,25), weighted means (21,22), inclusion filters (16,22), logarithmic transformation (19), and weighted correlation analysis (23). Outlier removal methods, for example using Dixon's test, assume a normal distribution for which there is little empirical basis.…”

“…The reporter ions are only liberated in MS/MS after the reporter ion and balance groups fragment from the labeled peptides during CID. iTRAQ has been applied to a wide range of biological applications from bacteria under nitrate stress (15) to mouse models of cerebellar dysfunction (16).…”

Extracellular Matrix Plasticity and GABAergic Inhibition of Prefrontal Cortex Pyramidal Cells Facilitates Relapse to Heroin Seeking

“…To identify relatively persistent heroin-induced changes in the synaptic proteome, the mPFC was dissected from the rats 21 days after the final heroin self-administration session (Figure 1b). Changes in a broad range of synaptic and synapse-associated proteins were quantified using isobaric Tags for Relative and Absolute Quantification (iTRAQ) combined with two-dimensional liquid chromatography and tandem mass spectrometry (2D LC-MS/MS), a powerful and reliable technology for quantification of neuronal proteins simultaneously from multiple samples (Hu et al, 2006;Li et al, 2007;Van den Oever et al, 2008). To isolate mPFC synaptic membrane fractions and to perform a duplicate measurement within a single iTRAQ experiment, two pools of heroin self-administration and two pools of saline self-administration (three animals per pool) were generated (Supplementary Figure S1).…”