Investigation of the ligand spectrum of human sterol carrier protein 2 using a direct mass spectrometry assay

Will A. Stanley; Kees Versluis; Carsten Schultz; Albert J.R. Heck; Matthias Wilmanns

doi:10.1016/j.abb.2007.02.026

Investigation of the ligand spectrum of human sterol carrier protein 2 using a direct mass spectrometry assay

Will A. Stanley
, Kees Versluis
, Carsten Schultz
, Albert J.R. Heck
, Matthias Wilmanns

Research output: Contribution to journal › Article › peer-review

8 Scopus citations

Abstract

Sterol carrier protein 2 (SCP2) has been investigated by nearly native electrospray ionisation mass spectrometry in the presence of long chain fatty acyl CoAs (LCFA-CoAs) and carnitine derivatives of equivalent fatty acid chain length (LCFA-carnitines). Four SCP2 constructs were compared to examine the influence of the N-terminal presequence and the C-terminal peroxisomal targeting signal on ligand binding. Removal of N- or C-terminal residues did not influence ligand binding. The observation that LCFA-CoAs are high affinity ligands for SCP2 was confirmed, while LCFA-carnitines were demonstrated for the first time not to interact with SCP2. LCFA-CoAs formed non-covalent complexes with SCP2 of 2:1 and 1:1 stoichiometry, which could be dissociated by elevating the energy of the ions upon entrance to the mass spectrometer. A fluorescence-competition assay using Nile Red butyric acid confirmed the mass spectrometric observations in solution. The physiological significance of the lack of LCFA-carnitine binding by SCP2 is discussed.

Original language	English (US)
Pages (from-to)	50-58
Number of pages	9
Journal	Archives of Biochemistry and Biophysics
Volume	461
Issue number	1
DOIs	https://doi.org/10.1016/j.abb.2007.02.026
State	Published - May 1 2007
Externally published	Yes

Funding

This work was partly supported by an EU Marie Curie Trainee Fellowship (HPMT-CT-2000-00045) to W.A.S. and EU Integrated Project on “Molecular Imaging” (LSHG-CT-2003-503259) to C.S. We thank Karel Wirtz, Ingrid Vereyken and Ben de Kruijff (Utrecht); Fabian Filipp and Michael Sattler (Heidelberg) for discussions and advice. Thanks also to Rajan Sankaranarayanan (Hyderabad) for constructive criticism of the manuscript and to Oliver Wichmann (Heidelberg) for synthesis of NR-BA.

Funders	Funder number
H2020 Marie Skłodowska-Curie Actions	HPMT-CT-2000-00045
European Commission	LSHG-CT-2003-503259

Keywords

Electrospray mass spectrometry
Fatty acyl CoA
Fatty acyl carnitine
Nile Red butyric acid
Non-specific lipid transfer protein
Sterol carrier protein 2 (SCP2)

ASJC Scopus subject areas

Biophysics
Biochemistry
Molecular Biology

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10.1016/j.abb.2007.02.026

Cite this

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title = "Investigation of the ligand spectrum of human sterol carrier protein 2 using a direct mass spectrometry assay",

abstract = "Sterol carrier protein 2 (SCP2) has been investigated by nearly native electrospray ionisation mass spectrometry in the presence of long chain fatty acyl CoAs (LCFA-CoAs) and carnitine derivatives of equivalent fatty acid chain length (LCFA-carnitines). Four SCP2 constructs were compared to examine the influence of the N-terminal presequence and the C-terminal peroxisomal targeting signal on ligand binding. Removal of N- or C-terminal residues did not influence ligand binding. The observation that LCFA-CoAs are high affinity ligands for SCP2 was confirmed, while LCFA-carnitines were demonstrated for the first time not to interact with SCP2. LCFA-CoAs formed non-covalent complexes with SCP2 of 2:1 and 1:1 stoichiometry, which could be dissociated by elevating the energy of the ions upon entrance to the mass spectrometer. A fluorescence-competition assay using Nile Red butyric acid confirmed the mass spectrometric observations in solution. The physiological significance of the lack of LCFA-carnitine binding by SCP2 is discussed.",

keywords = "Electrospray mass spectrometry, Fatty acyl CoA, Fatty acyl carnitine, Nile Red butyric acid, Non-specific lipid transfer protein, Sterol carrier protein 2 (SCP2)",

author = "Stanley, \{Will A.\} and Kees Versluis and Carsten Schultz and Heck, \{Albert J.R.\} and Matthias Wilmanns",

note = "Funding Information: This work was partly supported by an EU Marie Curie Trainee Fellowship (HPMT-CT-2000-00045) to W.A.S. and EU Integrated Project on “Molecular Imaging” (LSHG-CT-2003-503259) to C.S. We thank Karel Wirtz, Ingrid Vereyken and Ben de Kruijff (Utrecht); Fabian Filipp and Michael Sattler (Heidelberg) for discussions and advice. Thanks also to Rajan Sankaranarayanan (Hyderabad) for constructive criticism of the manuscript and to Oliver Wichmann (Heidelberg) for synthesis of NR-BA.",

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doi = "10.1016/j.abb.2007.02.026",

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AU - Stanley, Will A.

AU - Versluis, Kees

AU - Schultz, Carsten

AU - Heck, Albert J.R.

AU - Wilmanns, Matthias

N1 - Funding Information: This work was partly supported by an EU Marie Curie Trainee Fellowship (HPMT-CT-2000-00045) to W.A.S. and EU Integrated Project on “Molecular Imaging” (LSHG-CT-2003-503259) to C.S. We thank Karel Wirtz, Ingrid Vereyken and Ben de Kruijff (Utrecht); Fabian Filipp and Michael Sattler (Heidelberg) for discussions and advice. Thanks also to Rajan Sankaranarayanan (Hyderabad) for constructive criticism of the manuscript and to Oliver Wichmann (Heidelberg) for synthesis of NR-BA.

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N2 - Sterol carrier protein 2 (SCP2) has been investigated by nearly native electrospray ionisation mass spectrometry in the presence of long chain fatty acyl CoAs (LCFA-CoAs) and carnitine derivatives of equivalent fatty acid chain length (LCFA-carnitines). Four SCP2 constructs were compared to examine the influence of the N-terminal presequence and the C-terminal peroxisomal targeting signal on ligand binding. Removal of N- or C-terminal residues did not influence ligand binding. The observation that LCFA-CoAs are high affinity ligands for SCP2 was confirmed, while LCFA-carnitines were demonstrated for the first time not to interact with SCP2. LCFA-CoAs formed non-covalent complexes with SCP2 of 2:1 and 1:1 stoichiometry, which could be dissociated by elevating the energy of the ions upon entrance to the mass spectrometer. A fluorescence-competition assay using Nile Red butyric acid confirmed the mass spectrometric observations in solution. The physiological significance of the lack of LCFA-carnitine binding by SCP2 is discussed.

AB - Sterol carrier protein 2 (SCP2) has been investigated by nearly native electrospray ionisation mass spectrometry in the presence of long chain fatty acyl CoAs (LCFA-CoAs) and carnitine derivatives of equivalent fatty acid chain length (LCFA-carnitines). Four SCP2 constructs were compared to examine the influence of the N-terminal presequence and the C-terminal peroxisomal targeting signal on ligand binding. Removal of N- or C-terminal residues did not influence ligand binding. The observation that LCFA-CoAs are high affinity ligands for SCP2 was confirmed, while LCFA-carnitines were demonstrated for the first time not to interact with SCP2. LCFA-CoAs formed non-covalent complexes with SCP2 of 2:1 and 1:1 stoichiometry, which could be dissociated by elevating the energy of the ions upon entrance to the mass spectrometer. A fluorescence-competition assay using Nile Red butyric acid confirmed the mass spectrometric observations in solution. The physiological significance of the lack of LCFA-carnitine binding by SCP2 is discussed.

KW - Electrospray mass spectrometry

KW - Fatty acyl CoA

KW - Fatty acyl carnitine

KW - Nile Red butyric acid

KW - Non-specific lipid transfer protein

KW - Sterol carrier protein 2 (SCP2)

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SN - 0003-9861

VL - 461

SP - 50

EP - 58

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

IS - 1

ER -

Investigation of the ligand spectrum of human sterol carrier protein 2 using a direct mass spectrometry assay

Abstract

Funding

Keywords

ASJC Scopus subject areas

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