Biology Reference
In-Depth Information
Chapter 40
StageTip-Based HAMMOC, an Effi cient and Inexpensive
Phosphopeptide Enrichment Method for Plant Shotgun
Phosphoproteomics
Hirofumi Nakagami
Abstract
Phosphopeptide enrichment is the most critical step for successful LC-MS/MS-based shotgun phospho-
proteomics. Recent technological improvements have made selective phosphopeptide enrichment from
non-fractionated whole cell lysate digests with a single-step procedure possible. Here, a handy protocol is
described for phosphopeptide enrichment from plant materials using hydroxy acid-modifi ed metal oxide
chromatography (HAMMMOC) with a stop-and-go-extraction tip (StageTip).
Key words Shotgun phosphoproteomics, Plant, Phosphorylation, Phosphopeptide, HAMMOC,
Hydroxy acid-modifi ed metal oxide chromatography, Titania, StageTip, Stop-and-go-extraction tip
Abbreviations
ACN Acetonitrile
DTT Dithiothreitol
FASP Filter-aided sample preparation
HAMMOC Hydroxy acid-modifi ed metal oxide chromatography
IAM
Iodoacetamide
IMAC
Immobilized metal-ion affi nity chromatography
LC-MS
Liquid chromatography-mass spectrometry
LysC
Lysyl endopeptidase
MOC
Metal oxide chromatography
PTM
Posttranslational modifi cation
PTS
Phase-transfer surfactant
SCX
Strong cation exchange chromatography
StageTip
Stop-and-go-extraction tip
TFA
Trifl uoroacetic acid
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