High-Resolution PTMs Profiling Services

What Are Post-Translational Modifications (PTMs)?

DNA provides the blueprint for building proteins, but the true complexity of the proteome goes far beyond the genetic code. Once a gene is transcribed into mRNA and translated into a protein, additional chemical changes—called post-translational modifications (PTMs)—step in to fine-tune that protein's function, structure, and stability.

In mammalian cells, the genome contains around 25,000 genes. This gives rise to over 100,000 mRNA transcripts. Translation expands this into more than 100,000 proteins. But here's where it gets even more intricate: with the help of alternative splicing and PTMs, the number of distinct functional protein species can exceed 1 million. That's a 40-fold increase from gene to functional protein—a testament to how essential PTMs are in expanding protein diversity.

Many proteins, even after translation, are not fully active. Some require enzymatic cleavage, while others depend on chemical tweaks—PTMs—to reach their functional form. These modifications can dramatically alter a protein's behavior, from where it's located in the cell to how it interacts with other molecules.

Types of PTMs

PTMs involve covalent changes to proteins and fall into several main categories:

• Chemical group additions: These include phosphorylation, glycosylation, acylation, and alkylation—modifications that can impact protein folding, signalling, and activity.
• Attachment of small proteins or peptides: Examples include ubiquitination and SUMOylation, which often regulate protein degradation or nuclear transport.
• Structural alterations: Such as the formation of disulfide bonds, which help stabilize protein structure.

Even a single modification at a specific amino acid site can generate a protein with a completely new function. To date, scientists have identified more than 200 different types of PTMs across nearly every class of protein—from nuclear transcription factors and enzymes to membrane receptors and structural proteins.

This extraordinary biochemical flexibility allows cells to respond to internal and external cues with remarkable precision—something that's critical in areas like drug development, where even small changes in protein activity can influence therapeutic outcomes.

Figure 1. Cellular post-translational modifications^[1].

Comprehensive PTM Analysis Services by Creative Proteomics

Creative Proteomics offers end-to-end solutions for PTM proteomics analysis, leveraging years of expertise and state-of-the-art instrumentation. Whether you're studying protein regulation, signaling pathways, or disease mechanisms, our mass spectrometry-based PTM analysis services provide the precision and reliability you need.

Explore Our Broad PTM Profiling Capabilities

PTM Quantitative Proteomics Options

For researchers needing precise PTM quantification, we provide multiple labelling and label-free solutions:

• SILAC (Stable Isotope Labelling with Amino acids in Cell culture)
• iTRAQ/TMT (isobaric labelling for multiplexed quantification)
• label-free quantification, DIA (Data-Independent Acquisition), 4D-DIA label-free
• Targeted proteomics using SRM/PRM)

These technologies allow robust, reproducible measurements of PTM abundance across conditions or time points.

Download "Comparison of Three Label based Quantification Techniques iTRAQ TMT and SILAC"

MS-Based Strategies for Analysing PTMs

Protein function isn't determined by abundance alone. In fact, many key cellular activities depend on when, where, and how proteins are modified after translation. These PTMs—often reversible and highly dynamic—regulate protein activity, stability, localisation, and interactions.

To fully understand protein behaviour, it's essential to map PTMs across different biological contexts. That's where high-resolution mass spectrometry (MS) comes into play.

Why Mass Spectrometry Is Key to PTM Discovery

MS-based proteomics has transformed our ability to investigate PTMs with both breadth and precision. Here's why:

• Mass shift reveals modifications: When a protein undergoes a PTM, its molecular mass changes. MS instruments can detect these shifts with remarkable accuracy.
• High-throughput capability: Coupled with advanced search algorithms and bioinformatics platforms, MS enables large-scale identification of PTM sites across the proteome.
• Depth and versatility: Beyond PTMs, MS workflows also capture protein-protein interactions and subcellular localisation, offering a holistic view of cellular regulation.

In the past decade, this approach has revealed hundreds of thousands of PTM sites and uncovered previously unknown types of modifications in mammalian systems.

Enrichment Is Crucial for Low-Abundance PTMs

Despite MS sensitivity, many PTMs occur at low levels and fluctuate rapidly. This means that enrichment of modified peptides or proteins is often required before MS analysis. Without this step, rare or transient modifications may go undetected.

For example, using antibody-based enrichment or chemical tagging techniques significantly improves detection rates for modifications like phosphorylation or ubiquitination.

By combining sample enrichment with optimised MS workflows, researchers can now generate deep, quantitative insights into PTM landscapes—essential for uncovering regulatory mechanisms in cell biology, drug response, and disease progression.

Why Choose Creative Proteomics for MS-Based PTM Analysis?

1. High Accuracy with Low Variability

Our advanced MS systems and validated enrichment strategies yield precise, reproducible results with <5% coefficient of variation.

2. Versatile Sample Compatibility

We work with diverse sample types—from microbial cultures and cell lines to tissues and complex fluids—ensuring wide research applicability.

3. Expert Support at Every Step

With a seasoned PTM proteomics team and strict quality control, we deliver actionable, high-confidence data you can trust.

Step-by-Step Workflow for Identifying and Quantifying PTMs

Identifying post-translational modifications (PTMs) is key to understanding how proteins behave in different biological systems. Whether you're optimising a mass spectrometry-based PTM analysis or developing new monoclonal antibody production tips, a well-structured experimental workflow is critical for accurate results.

Here's how a typical PTM analysis pipeline unfolds:

1. The experiment designed (clear intention);
2. Choice of MS instrument and analysis method;
3. Methods optimization;
4. Sample preparation-protein extraction and determination of concentration;
5. Proper protease(s) for full proteolytic digestion;
6. PTM-peptides enrichment (optional);
7. Peptides fractionations for in-depth identification by reversed phase high performance liquid chromatography (optional);
8. LC-MS/MS analysis;
9. Bioinformation analysis for full PTM proteins and sites annotation.

For a deeper dive into the science behind PTM workflows, don't miss our expert resource: [Overview of Post-Translational Modification Analysis].

Top-tier Platform

Equipped with top-tier platforms like Q-Exactive, Q-Exactive HF, and Orbitrap Fusion™ Tribrid™,timsTOF Pro mass spectrometer, we ensure fast, sensitive, and scalable results with reliable reproducibility across batches.

Thermo Q Exactive^TM series

AB Sciex 6500+

Thermo Orbitrap Fusion Lumos

Bruker timsTOF Pro

Application Scenarios

PTM analysis is essential in studies requiring precise regulation of protein function, including:

• Drug Target Validation: Identify functional PTM sites critical for target activation or inhibition.
• Cancer Mechanism Research: Uncover dysregulated PTMs driving tumor progression or resistance.
• Signal Transduction Studies: Map dynamic phosphorylation or acetylation patterns in response to stimuli.
• Metabolic Pathway Analysis: Link acylation or succinylation changes to metabolic disorders.
• Neurodegeneration and Aging: Profile ubiquitination or SUMOylation events associated with protein turnover.

Whether for biomarker discovery or therapeutic development, our PTM profiling empowers high-confidence, actionable insights.

Sample Requirement

Deliverables

• A report that details:
• Types of post-translational modifications
• PTM locations and frequency
• Sample and method details
• A summary of quantitative PTM data
• An Excel file or HTML report with:
• Complete qualitative and quantitative PTM data
• Statistical analysis of modification changes across samples

Frequently Asked Questions (FAQ) on PTM Proteomics

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Customer Case Study

Phosphorylation Site Mapping of TcPolβ Reveals Kinase-Specific Regulation inTrypanosoma cruzi

Published In: Microorganisms (2024)
Client: Universidad de Chile / Universidad de Valencia Research Collaboration
Service Provided by Creative Proteomics: Phospho-peptide enrichment & site-specific phosphorylation analysis by LC-MS/MS

DOI: https://doi.org/10.3390/microorganisms12050907

• Project Background
• How Creative Proteomics Helped
• Key Results Enabled by Our Platform

DNA polymerase β (TcPolβ) in T. cruzi is essential for kinetoplast DNA repair and replication—processes vital for parasite survival. However, the molecular regulation of TcPolβ remained unclear. The research team sought to systematically characterize how different protein kinases modulate TcPolβ activity via phosphorylation and whether activation pathways (e.g., PKC-dependent signaling) enhance this modification in vitro and in vivo.

To resolve kinase-specific phosphorylation patterns, the researchers entrusted Creative Proteomics with the downstream phosphoproteomics workflow:

Step	Details
Sample Type	Recombinant TcPolβ phosphorylated in vitro by 4 kinases (TcCK1, TcCK2, TcAUK1, TcPKC1)
Sample Prep	In-gel digestion of TcPolβ bands from SDS-PAGE
Enrichment	Phosphopeptide recovery from trypsin-digested gel bands
Platform	NanoLC-MS/MS on high-resolution instruments
Bioinformatics	MaxQuant-based phosphosite localization and sequence alignment (using TcPolβ reference RNC61524.1)

Advantage: Enabled detection of both serine/threonine and rare tyrosine phosphorylation events across specific kinase conditions.

Site-level Resolution:

MS analysis revealed 25 phosphorylation sites across all kinase treatments:

• TcCK1: 12 sites
• TcCK2 & TcAUK1: 3 sites each

TcPKC1: 7 sites

Table 1. Phosphorylated residues on TcPolβ by the indicated protein kinases as determined by MS analysis.

Kinase	Phosphorylated Residues on TcPolβ
AUK1	S 69	S 275	T 366
CK2	S 69	S 275	T 382
CK1	S 13	S 46	T 49	S 69
	S 185	S 193	S 275	T 278
	Y 307	S 336	T 338	T 345
	T 382
PKC1	S 13	S 46	T 49	S 69
	S 185	S 275	T 382	Y 302

Unexpected Dual-Specificity:

Western blot analysis of TcPolβ phosphorylation by different T. cruzi protein kinases and detected by anti-phosphor antibodies.

Publications

Here are some publications in Proteomics research from our clients:

References

1. Jensen ON. Interpreting the protein language using proteomics. Nature Reviews Molecular Cell Biology. 2006 Jun;7(6):391-403. https://doi.org/10.1038/nrm1939
2. Mann M, Jensen ON. Proteomic analysis of post-translational modifications. Nature Biotechnology. 2003 Mar;21(3):255-61. https://doi.org/10.1038/nbt0303-255
3. Brandi J, Noberini R, Bonaldi T, et al. Advances in enrichment methods for mass spectrometry-based proteomics analysis of post-translational modifications. Journal of Chromatography A. 2022 Aug 16;1678:463352. DOI: 10.1016/j.chroma.2022.463352