Quantitative multiple reaction monitoring analysis of synaptic proteins from human brain.

BACKGROUND

The recent introduction of multiple reaction monitoring to proteomics research has allowed many researchers to apply this technique to study human diseases.

NEW METHODS

Here we combine subcellular fractionation of human autopsy brain with label-free multiple reaction monitoring to quantitatively analyse proteins in synapses. The protein enolase, from Streptococcus pyogenes serotype M6, which is sufficiently different from human proteins, was spiked into the sample mixture prior to trypsin digestion and used as an internal standard across samples.

RESULTS

Three synaptic proteins and an internal standard analysed with four injections over four consecutive days gave consistent differences with a coefficient of variation of <4%. Consistent retention time was recorded across the replicates. Comparison with existing methods: Previously, multiple reaction monitoring analysis has been utilized to study human autopsy and animal tissues. Utilizing the synaptosomal fraction prior to analysis reduced sample complexity and allowed the enriched synaptic proteins to be quantitatively assessed in a highly reproducible manner, without the need for expensive fluorescent labels and synthetic peptides.

CONCLUSION

Protein expression can be measured with accuracy using label-free multiple reaction monitoring mass spectrometry in relatively complex human brain samples. Synaptic functions are critical for neuronal communication and function, and synapse dysfunction underlies many neurodegenerative diseases, including Alzheimer's disease. This method can be applied to study a range of brain disorders.