Available online at www.sciencedirect.com
Journal of Chromatography A, 1172 (2007) 57–71
Fe
3
O
4
@Al
2
O
3
magnetic core–shell microspheres for rapid and highly
specific capture of phosphopeptides with mass spectrometry analysis
Yan Li
a
, Yingchao Liu
b
, Jia Tang
a
, Huaqing Lin
a
, Ning Yao
a
, Xizhong Shen
c
,
Chunhui Deng
a,∗
, Pengyuan Yang
a
, Xiangmin Zhang
a,∗∗
a
Department of Chemistry & Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China
b
Shanghai Neurosurgical Center, Department of Neurosurgery, Huashan Hospital, Shanghai Medical College,
Fudan University, Shanghai 200040, China
c
Zhongshan Hospital, Shanghai Medical College, Fudan University, Shanghai 200400, China
Received 23 May 2007; received in revised form 20 September 2007; accepted 26 September 2007
Available online 2 October 2007
Abstract
Selective detection of phosphopeptides from complex biological samples is a challenging and highly relevant task in many proteomics applica-
tions. In this study, a novel phosphopeptide enrichment approach based on the strong interaction of Fe
3
O
4
@Al
2
O
3
magnetic core–shell microspheres
with phosphopeptides has been developed. With a well-defined core–shell structure, the Fe
3
O
4
@Al
2
O
3
magnetic core–shell microspheres not only
have a shell of aluminum oxide, giving them a high-trapping capacity for the phosphopeptides, but also have magnetic property that enables easy
isolation by positioning an external magnetic field. The prepared Fe
3
O
4
@Al
2
O
3
magnetic core–shell microspheres have been successfully applied
to the enrichment of phosphopeptides from the tryptic digest of standard phosphoproteins -casein and ovalbumin. The excellent selectivity of this
approach was demonstrated by analyzing phosphopeptides in the digest mixture of -casein and bovine serum albumin with molar ratio of 1:50 as
well as tryptic digest product of casein and five protein mixtures. The results also proved a stronger selective ability of Fe
3
O
4
@Al
2
O
3
magnetic
core–shell microspheres over Fe
3+
-immobilized magnetic silica microspheres, commercial Fe
3+
–IMAC (immobilized metal affinity chromatog-
raphy) resin, and TiO
2
beads. Finally, the Al
2
O
3
coated Fe
3
O
4
microspheres were successfully utilized for enrichment of phosphopeptides from
digestion products of rat liver extract. These results show that Fe
3
O
4
@Al
2
O
3
magnetic core–shell microspheres are very good materials for rapid
and selective separation and enrichment of phosphopeptides.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Separation and enrichment; Phosphorylated peptide; Alumina; Magnetic microsphere; MALDI–TOF-MS
1. Introduction
The reversible phosphorylation of proteins catalyzed by
kinases and phosphatases is a general mechanism for fine-
tuning protein structure and function. Protein phosphorylation
plays a key role in the regulation of important cellu-
lar functions such as cell growth, cell differentiation, and
metabolism [1–4]. Mass spectrometry, including electrospray
ionisation–mass spectrometry (ESI–MS) and matrix-assisted
laser desorption/ionisation–mass spectrometry (MALDI–MS),
is an important technology for the detection and charac-
∗
Corresponding author. Tel.: +86 21 6564 3983; fax: +86 21 6564 1740.
∗∗
Corresponding author.
E-mail addresses: chdeng@fudan.edu.cn (C. Deng),
xmzhang@fudan.edu.cn (X. Zhang).
terization of protein phosphorylation and phosphopeptides
[5–7]. However, the identification and characterization of phos-
phoprotein by mass spectrometry is still one of the most
challenging tasks in contemporary proteome research because
of two reasons. First, although a large percentage of cellu-
lar proteins can be phosphorylated [8,9], the abundance of
the individual phosphorylated forms is frequently low. Sec-
ond, the signals of the phosphopeptides are often suppressed
by nonphosphorylated-peptide residues contained in the protein
digest. Without specific enrichment, only the most abun-
dant phosphoproteins would be identified. Therefore, selective
detection of phosphopeptides from proteolytic digests is a
challenging and highly relevant task in many proteomics appli-
cations.
Specific capture of phosphopeptides is possible with several
strategies, including the addition of an affinity tag to phos-
0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2007.09.062