Overview of Post-translational Modifications (PTMs) Analysis
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Phosphorylation: Occurs predominantly on serine (Ser), threonine (Thr), and tyrosine (Tyr) residues, and serves as a key regulatory mechanism in signal transduction pathways;
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Acetylation: Commonly modifies lysine residues and plays a crucial role in chromatin remodeling and transcriptional regulation;
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Ubiquitination: Involves the attachment of polyubiquitin chains to substrate proteins, thereby mediating protein degradation or functional modulation;
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Methylation: Influences gene expression and contributes to the regulation of epigenetic states;
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Glycosylation: Essential for cell–cell recognition, protein stability, and immune modulation;
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Other modifications such as nitration, hydroxylation, palmitoylation, and sulfation: Involved in diverse regulatory processes including mitochondrial function, receptor activation, and membrane anchoring of proteins.
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Phosphorylated peptides can be enriched using TiO₂ or Fe-NTA affinity columns;
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Ubiquitinated residues are commonly recognized by K-ε-GG–specific antibodies;
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Acetylated peptides are enriched via immunoprecipitation or strong cation exchange chromatography (SCX).
Proteins play fundamental roles in virtually all biological processes, from signal transduction to the maintenance of cellular architecture. However, newly synthesized proteins are not functionally active immediately following translation; they typically require a series of post-translational modifications (PTMs) to become functionally competent, properly localized within subcellular compartments, or targeted for degradation. These modifications endow proteins with regulatory complexity and serve as essential mechanisms of cellular fine-tuning. Comprehensive analysis of PTMs is not only central to basic life science research, but also represents a key strategy for uncovering disease mechanisms and identifying potential therapeutic targets.
Diversity and Functional Significance of Post-translational Modifications
Post-translational modifications refer to a wide array of molecular processes that introduce chemical groups or induce structural changes in proteins after their synthesis, via enzymatic or non-enzymatic mechanisms. To date, over 400 distinct types of PTMs have been characterized. Among the most prevalent are:
These PTMs are conserved across a broad spectrum of organisms—including mammals, plants, and microorganisms—and participate in the regulation of numerous cellular processes such as the cell cycle, metabolic homeostasis, stress responses, and various pathological conditions (e.g., cancer, autoimmune diseases, and neurodegenerative disorders).
Analytical Strategies for Post-Translational Modifications (PTMs)
At present, mass spectrometry (MS) has become the central platform for PTM investigations. High-resolution MS-based proteomic approaches enable the high-throughput and quantitative identification of global modification sites without the need for antibodies. The general workflow typically involves the following steps:
1. Protein Extraction and Enzymatic Digestion
Proteins in complex samples are typically digested with trypsin to generate peptides and expose potential modification sites. To maximize the recovery of modified peptides, lysis conditions should be carefully optimized to minimize modification loss.
2. Enrichment of Modified Peptides
Given the inherently low abundance and weak signals of modified peptides, targeted enrichment strategies are essential prior to MS analysis. For instance:
3. Liquid Chromatography–Mass Spectrometry (LC-MS/MS)
Nano-flow liquid chromatography (nanoLC) coupled with high-resolution MS instruments such as Orbitrap or Q Exactive allows efficient separation and detection of modified peptides. Tandem MS (MS/MS) provides detailed structural information of peptide fragments, enabling precise localization of modification sites.
4. Data Analysis and Annotation
Modified peptides are identified using dedicated software tools such as MaxQuant and Proteome Discoverer, in conjunction with the Unimod database for modification matching. To ensure data reliability, researchers apply stringent false discovery rate (FDR) thresholds and validate results through manual inspection and biological replicates.
Highlights in Specific Types of Modifications
1. Phosphoproteomics
Phosphorylation is among the most extensively studied PTMs and plays a critical role in signal transduction. High-throughput phosphoproteomic analyses have successfully identified activation sites in key pathways, such as EGFR and MAPK, with broad applications in cancer target discovery.
2. Ubiquitination and SUMOylation
The study of ubiquitination has expanded beyond its classical role in protein degradation to encompass functions in signal regulation and cellular stress responses. Recently, small ubiquitin-like modifiers (SUMO) have gained attention for their involvement in DNA repair and viral infection, marking a new frontier in protein homeostasis research.
3. Glycosylation and Immune-related Mechanisms
Glycosylation plays a pivotal role in cell recognition and immune evasion. Abnormal N- or O-linked glycosylation patterns are closely associated with cancer immune suppression. The integration of MS with glycan profiling supports the optimization of therapeutic antibodies and the purification of biopharmaceuticals.
PTM research is transitioning from a complementary tool for protein functional annotation to a core component of cellular regulatory studies. It is now indispensable for elucidating complex biological processes. Whether in pathway mapping, biomarker discovery, or drug target identification, PTM analysis provides critical and unique insights. To enable efficient PTM studies, robust experimental platforms must be complemented by standardized and systematic technical services. With extensive experience in MS and comprehensive proteomics solutions, MtoZ Biolabs offers high-quality PTM proteomic analysis services, supporting the elucidation of cellular regulatory mechanisms and accelerating the translation of scientific discoveries.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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