Phosphosite Mapping of HIP-55 Protein in Mammalian Cells

Phosphosite Mapping of HIP-55 Protein in Mammalian Cells

In the present study, hematopoietic progenitor kinase 1 (HPK1)-interacting protein of 55 kDa (HIP-55) protein was over-expressed in HEK293 cells, which was genetically attached with 6x His tag. The protein was purified by nickel-charged resin and was then subjected to tryptic digestion. The phosphor...

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Main Authors: Ning Liu, Ningning Sun, Xiang Gao, Zijian Li
Format: Article
Language:English
Published: MDPI AG 2014-03-01
Series:International Journal of Molecular Sciences
Subjects:
mass spectrometry
HIP-55
phosphorylation
Online Access:http://www.mdpi.com/1422-0067/15/3/4903
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spelling doaj-dbca00a2a4bf404f85e7606178ad4ed32020-11-24T23:41:09ZengMDPI AGInternational Journal of Molecular Sciences1422-00672014-03-011534903491410.3390/ijms15034903ijms15034903Phosphosite Mapping of HIP-55 Protein in Mammalian CellsNing Liu0Ningning Sun1Xiang Gao2Zijian Li3Central Laboratory, Jilin University Second Hospital, Changchun 130041, ChinaCentral Laboratory, Jilin University Second Hospital, Changchun 130041, ChinaCentral Laboratory, Jilin University Second Hospital, Changchun 130041, ChinaInstitute of Vascular Medicine, Peking University Third Hospital, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides, Ministry of Health, Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education and Beijing Key Laboratory of Cardiovascular Receptors Research, Beijing 100191, ChinaIn the present study, hematopoietic progenitor kinase 1 (HPK1)-interacting protein of 55 kDa (HIP-55) protein was over-expressed in HEK293 cells, which was genetically attached with 6x His tag. The protein was purified by nickel-charged resin and was then subjected to tryptic digestion. The phosphorylated peptides within the HIP-55 protein were enriched by TiO2 affinity chromatography, followed by mass spectrometry analysis. Fourteen phosphorylation sites along the primary structure of HIP-55 protein were identified, most of which had not been previously reported. Our results indicate that bio-mass spectrometry coupled with manual interpretation can be used to successfully identify the phosphorylation modification in HIP-55 protein in HEK293 cells.http://www.mdpi.com/1422-0067/15/3/4903mass spectrometryHIP-55phosphorylation
collection DOAJ
language English
format Article
sources DOAJ
author Ning Liu
Ningning Sun
Xiang Gao
Zijian Li
spellingShingle Ning Liu
Ningning Sun
Xiang Gao
Zijian Li
Phosphosite Mapping of HIP-55 Protein in Mammalian Cells
International Journal of Molecular Sciences
mass spectrometry
HIP-55
phosphorylation
author_facet Ning Liu
Ningning Sun
Xiang Gao
Zijian Li
author_sort Ning Liu
title Phosphosite Mapping of HIP-55 Protein in Mammalian Cells
title_short Phosphosite Mapping of HIP-55 Protein in Mammalian Cells
title_full Phosphosite Mapping of HIP-55 Protein in Mammalian Cells
title_fullStr Phosphosite Mapping of HIP-55 Protein in Mammalian Cells
title_full_unstemmed Phosphosite Mapping of HIP-55 Protein in Mammalian Cells
title_sort phosphosite mapping of hip-55 protein in mammalian cells
publisher MDPI AG
series International Journal of Molecular Sciences
issn 1422-0067
publishDate 2014-03-01
description In the present study, hematopoietic progenitor kinase 1 (HPK1)-interacting protein of 55 kDa (HIP-55) protein was over-expressed in HEK293 cells, which was genetically attached with 6x His tag. The protein was purified by nickel-charged resin and was then subjected to tryptic digestion. The phosphorylated peptides within the HIP-55 protein were enriched by TiO2 affinity chromatography, followed by mass spectrometry analysis. Fourteen phosphorylation sites along the primary structure of HIP-55 protein were identified, most of which had not been previously reported. Our results indicate that bio-mass spectrometry coupled with manual interpretation can be used to successfully identify the phosphorylation modification in HIP-55 protein in HEK293 cells.
topic mass spectrometry
HIP-55
phosphorylation
url http://www.mdpi.com/1422-0067/15/3/4903
work_keys_str_mv AT ningliu phosphositemappingofhip55proteininmammaliancells
AT ningningsun phosphositemappingofhip55proteininmammaliancells
AT xianggao phosphositemappingofhip55proteininmammaliancells
AT zijianli phosphositemappingofhip55proteininmammaliancells
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