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Fast and easy phosphopeptide fractionation by combinatorial ERLIC-SCX solid-phase extraction for in-depth phosphoproteome analysis.

Abstract：

:Mass spectrometry-based phosphoproteomic analysis is a powerful method for gaining a global, unbiased understanding of cellular signaling. Its accuracy and comprehensiveness stands or falls with the quality and choice of the applied phosphopeptide prefractionation strategy. This protocol covers a powerful but simple and rapid strategy for phosphopeptide prefractionation. The combinatorial use of two distinct chromatographic techniques that address the inverse physicochemical properties of peptides allows for superior fractionation efficiency of multiple phosphorylated peptides. In the first step, multiphosphorylated peptides are separated according to the number of negatively charged phosphosites by electrostatic repulsion-hydrophilic interaction chromatography (ERLIC). A subsequent strong cation exchange (SCX) step separates mostly singly phosphorylated peptides in the ERLIC flow-through according to their positive charge. The presented strategy is inexpensive and adaptable to large and small amounts of starting material, and it allows highly multiplexed sample preparation. Because of its implementation as solid-phase extraction, the entire workflow takes only 2 h to complete.

journal_name

Nat Protoc

journal_title

Nature protocols

authors

Zarei M,Sprenger A,Rackiewicz M,Dengjel J

doi

10.1038/nprot.2015.134

subject

Has Abstract

pub_date

2016-01-01 00:00:00

pages

37-45

issue

1

eissn

1754-2189

issn

1750-2799

pii

nprot.2015.134

journal_volume

11

pub_type

杂志文章

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