Characterization of the Phosphoproteome and Sialoproteome in Brain Tissues by Mass Spectrometry

  • María Ibáñez-Vea
  • Stefan J. Kempf
  • Martin R. Larsen
Protocol
Part of the Neuromethods book series (NM, volume 127)

Abstract

Mass spectrometry is an essential tool for the characterization of proteins within neuroscience. The development of faster instruments enables neuroscientists to investigate a large proportion of the proteome in the brain in only short analysis time. Yet, a detailed functional investigation of the intrinsic biochemical processes of the brain by evaluation of the post-translational modifications in proteins is still missing. Phosphorylation and N-linked glycosylation are important protein modifications within the brain as they are involved in neural development, neurotransmission, neurite extension, and synaptic plasticity. Although the importance of these protein modifications is undoubtable for the brain functionality, only a few global protein modification datasets have been generated so far in the neuroscience field. This is due to the higher difficulties to sensitively and specifically enrich these low abundant protein modifications from the high abundant nonmodified peptides and from the very lipid-rich brain material.

Here, we describe how a highly selective, sensitive, low hands-on-time and cost-effective simultaneous enrichment of phosphorylated peptides, sialylated N-linked glycopeptides as well as intact sialylated N-linked glycopeptides and unmodified peptides from the same biological sample can be applied to bridge this gap in neuroscience, exemplified by a proteomic characterization of the murine brain growth spurt.

Key words

Alzheimer’s disease Phosphoproteomics Sialylated N-linked glycoproteomics Synaptic plasticity 

Abbreviation

AAA

Amino acid analysis

ACN

Acetonitrile

AMPA

Alpha-amino-2-hydroxy-5-methyl-4-isoxazole propionic acid receptor

CDG

Congenital disorder of glycosylation

CNS

Central nervous system

DTT

Dithiothreitol

HCD

Higher collisional-induced dissociation

HILIC

Hydrophilic interaction liquid chromatography

IAA

Iodoacetamide

IMAC

Iron metal affinity chromatography

LTD

Long-term depression

LTP

Long-term potentiation

MALDI

Matrix-assisted laser desorption ionization

MeOH

Methanol

mGluR

Metabotropic G-protein-coupled glutamate receptor

MS

Mass spectrometry

nLC-MS/MS

Nano liquid chromatography-Tandem mass spectrometry

NMDAR

N-Methyl-d-aspartate receptor

PTM

Post-translational modification

RT

Room temperature

SIMAC

Sequential elution from IMAC

SPE

Solid phase extraction

TEAB

Triethylammonium bicarbonate

TFA

Trifluoroacetic acid

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • María Ibáñez-Vea
    • 1
  • Stefan J. Kempf
    • 2
  • Martin R. Larsen
    • 2
  1. 1.Immunomodulation Group, Navarrabiomed Biomedical Research CenterNavarra Institute for Health Research (IdiSNA)PamplonaSpain
  2. 2.Department of Biochemistry and Molecular BiologyUniversity of Southern DenmarkOdenseDenmark

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