ABSTRACT
Post-translational modifications (PTMs) impart structural heterogeneities that can alter plasma proteins' functions in various pathophysiological processes. However, the identification and mapping of PTMs in untargeted plasma proteomics is still a challenge due to the presence of diverse components in blood. Here, we report a label-free method for identifying and mapping hydroxylated proteins using tandem mass spectrometry (MS/MS) in the human plasma sample. Our untargeted proteomics approach led us to identify 676 de novo sequenced peptides in human plasma that correspond to 201 proteins, out of which 11 plasma proteins were found to be hydroxylated. Among these hydroxylated proteins, Immunoglobulin A1 (IgA1) heavy chain was found to be modified at residue 285 (Pro285 to Hyp285), which was further validated by MS/MS study. Molecular dynamics (MD) simulation analysis demonstrated that this proline hydroxylation in IgA1 caused both local and global structural changes. Overall, this study provides a comprehensive understanding of the protein profile containing Hyp PTMs in human plasma and shows the future perspective of identifying and discriminating Hyp PTM in the normal and the diseased proteomes.
Subject(s)
Blood Proteins , Hydroxyproline , Protein Processing, Post-Translational , Proteome , Proteomics , Blood Proteins/analysis , Blood Proteins/metabolism , Chromatography, Liquid , Humans , Hydroxyproline/analysis , Hydroxyproline/metabolism , Proteome/analysis , Proteome/metabolism , Tandem Mass SpectrometryABSTRACT
AIMS: Diagnostic ambiguities regarding the malignant potentiality of oral submucous fibrosis (OSF), an oral precancerous condition having dysplastic and non-dysplastic isoforms are the major failure for early intervention of oral squamous cell carcinoma (OSCC) patients. Our goal is to identify proteomic signatures from biopsies that can be used as precancer diagnostic marker for patient suffering from OSF. METHODS: The high throughput techniques adopting de novo peptide sequencing (1D SDS-PAGE coupled nanoLC MALDI tandem mass spectrometry (MS/MS)-based peptide mass fingerprint), immunohistochemistry (IHC), Western blot (WB) and real-time PCR (RT-PCR) analysis are considered for such biomarker identification and multilevel validations. RESULTS: Alpha-enolase is identified as an overexpressed protein in biopsies of oral submucous fibrosis with dysplasia (OSFWD) compared with oral submucous fibrosis without dysplasia (OSFWT) and normal oral mucosa (NOM). Total proteome analysis of an overexpressed protein band around 47 kDa of OSFWD identifies 334 peptides corresponding to 61 human proteins. Among them α-enolase is identified as a prime protein with highest number of peptides (44 out of 334 peptides) and sequence coverage (66.4%). Furthermore, RT-PCR, WB and IHC analysis also show mRNA and tissue level upregulation of α-enolase in OSFWD validating α-enolase as precancer marker. CONCLUSIONS: This study for the first time identifies and validates α-enolase as a novel biomarker for early diagnosis of malignant potentiality of OSF. Hence, the identified protein marker, α-enolase can help in early therapeutic intervention of OSF patients leading to the reduction of patient's pain, treatment cost and enhancement of patient's quality of life.
