High-Resolution PTMs Profiling Services

Creative Proteomics provides comprehensive, high-resolution PTM profiling services using cutting-edge mass spectrometry platforms. With extensive experience in protein research, we offer customized solutions for a wide spectrum of post-translational modifications—supporting studies in cell signaling, disease mechanisms, metabolic regulation, and more.

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  • Background
  • Service Overview
  • Methods
  • Advantage
  • Workflow
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  • Sample Requirement
  • FAQ
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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.

Glycolysis Pathway Analysis ServiceFigure 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

Service Contents

  • Common PTMs
  • Emerging and Specialized PTMs
  • Histone and Chromatin Modifications
  • Other PTMs Services

Common PTMs

Phosphorylation Analysis

Identify and quantify phosphorylation sites to study signaling pathways.

Acetylation Analysis

Examine acetylation modifications affecting protein function and gene expression.

Methylation Analysis

Detect methylation patterns influencing protein interactions and stability.

Ubiquitination Analysis

Analyze ubiquitin attachments that regulate protein degradation.

Glycosylation Analysis

Characterize glycan structures impacting protein folding and cell signaling.

SUMOylation Analysis

Investigate SUMO protein conjugation affecting nuclear transport and transcriptional regulation.

Emerging and Specialized PTMs

Crotonylation Analysis

Explore crotonylation's role in gene regulation and chromatin remodeling.

S-Nitrosylation Analysis

Assess nitric oxide-mediated modifications influencing protein function.

Lipidation Analysis

Study lipid attachments that target proteins to membranes.

Hydroxylation Site Identification

Identify hydroxyl groups added to amino acids, affecting protein stability.

Biotinylation Analysis

Detect biotin attachments used in protein purification and labeling.

Succinylation Analysis

Examine succinyl groups modifying lysine residues, impacting metabolism.

Propionylation Analysis

Analyze propionyl modifications influencing chromatin structure.

Acylation Analysis

Investigate acyl group additions affecting protein localization and function.

Alkylation Analysis

Study alkyl group additions that can alter protein activity.

Glutathione Site Identification

Identify glutathione conjugation sites involved in oxidative stress responses.

Glutamylation Analysis

Examine glutamyl groups added to proteins, affecting microtubule functions.

S-Prenylation Analysis

Analyze prenyl groups facilitating membrane attachment of proteins.

S-Myristoylation Analysis

Detect myristoyl groups influencing protein-membrane interactions.

Palmitoylation Analysis

Study palmitoyl modifications affecting protein trafficking.

Di-Sulfide Bond Localization

Map disulfide bonds critical for protein structure and stability.

Protein Glycation Analysis

Assess non-enzymatic sugar additions linked to aging and diabetes.

Histone and Chromatin Modifications

Histone PTMs Analysis

Comprehensive profiling of histone modifications influencing gene expression.

Methyl-Proteomics

Detailed analysis of methylation across the proteome.

Acetyl-Proteomics

In-depth study of acetylation patterns affecting chromatin dynamics.

Other PTMs Services

Customized analysis of additional or less common PTMs as per research requirements.

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.

PTM profiling workflows

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 ExactiveTM series

Thermo Q ExactiveTM series

AB Sciex 6500+

AB Sciex 6500+

Thermo Orbitrap Fusion Lumos

Thermo Orbitrap Fusion Lumos

Bruker timsTOF Pro

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

Sample typeRecommended sample size
Animal tissuesHard tissues (bones, hair)300-500mg
Soft tissues (leaves, flowers of woody plants, herbaceous plants, algae, ferns)200mg
Plant tissuesHard tissues (roots, bark, branches, seeds, etc.)3-5g
MicrobesCommon bacteria, fungal cells (cell pellets)100μL
cellsSuspension/adherent cultured cells (cell count/pellet)>1*107
FluidsPlasma/serum/cerebrospinal fluid (without depletion of high abundance proteins)20μL
Plasma/serum/cerebrospinal fluid (with depletion of high abundance proteins)100μL
Follicular fluid200μL
Lymph, synovial fluid, puncture fluid, ascites5mL
OthersSaliva/tears/milk3-5mL
Culture supernatant (serum-free medium cannot be used)20mL
Pure protein (best buffer is 8MUrea)300μg
FFPEEach slice: 10µm thickness, 1.5×2cm area15-20 slices

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

What Are the Most Common Post-Translational Modifications—and How Do They Differ?

Common Post-Translational Modifications (PTMs) – Summary Table

PTM TypeΔMass (Da)MS/MS StabilityTarget Site(s)Enrichment MethodBiological Roles & Applications
Phosphorylation+80+++ (Tyr), +/+ (Ser/Thr)Ser, Thr, TyrTiO₂, IMAC-Fe, Phospho-specific antibodiesSignal transduction, stress response, cell cycle, cancer pathways
Acetylation+42+++LysAcetyl-lysine antibodiesHistone modification, transcription regulation, apoptosis, protein stability
Methylation+14+++Arg, LysMethylation-specific antibodiesEpigenetic regulation, protein trafficking, signal transduction
Ubiquitination>1,000+/+LysUbiquitin-specific antibodiesProtein degradation, cell cycle control, neurodegenerative research
Succinylation+100–115++LysSuccinylation-specific antibodiesMetabolic regulation, inflammation, epigenetic remodeling
N-Glycosylation>800+/+AsnHILICProtein secretion, immune response, cell recognition
O-Glycosylation203–>800+/+Ser, ThrHILICSignaling, disease biomarkers, stress adaptation
Fatty Acylation (Farnesyl, Myristoyl, Palmitoyl)+204 to +238+++ to +/+Cys, Gly, Ser (varies)Affinity purification, resin trappingMembrane tethering, localization, protein-protein interactions
GPI Anchoring>1,000++C-terminal signalBiotin-labeling, detergent phase separationAnchoring proteins to outer plasma membrane
Hydroxylation+16+++Pro, LysStandard peptide enrichmentCollagen stability, protein-ligand binding
Sulfation (sTyr)+80+TyrAnion exchange, antibodiesReceptor-ligand interactions, protein trafficking
Disulfide Bonds-2++Cys-CysReduction-alkylation strategiesProtein folding, structural stability
Deamidation+1+++Asn, GlnAcidic hydrolysis artifactsRegulatory effect, common modification artifact
Pyroglutamylation-17+++N-terminal Gln/GluEnzyme-based cleavage detectionBlocks N-terminus, increases protein stability
Tyrosine Nitration+45+/+TyrNitrotyrosine antibodiesOxidative stress marker, inflammation

ΔMass (Da): Mass shift upon modification

MS/MS Stability: + (labile), ++ (moderately stable), +++ (stable)

Enrichment Method: Key to targeted MS analysis

Biological Roles: Summarized based on current research relevance in proteomics, drug development, and cell signaling

Why is PTM peptide enrichment important for mass spectrometry-based analysis?

Enriching PTM peptides before mass spectrometry (MS) greatly improves sensitivity and accuracy. This is especially crucial for detecting low-abundance or dynamic modifications that may be missed in direct MS analysis.

How do I choose the right enrichment method for my PTM analysis?

The choice depends on:

  • The type of post-translational modification (PTM)
  • The specific amino acid residues modified
  • The goal of your experiment (e.g., broad profiling vs. targeted analysis)
    Matching your method to these factors ensures better identification and quantification results.

What are the best enrichment strategies for PTMs?

Protein PTMsTarget amino acid residuesAffinity enrichment strategiesChemical enrichment strategies
PhosphorylationSer, Thr, Tyr, His, Arg, Lys, Asp, Cys, GluIMAC, MOAC, SIMAC, SIMAC-HILIC, SCX, SAX, immunoenrichment, superbinder SH2 domains, ERLIC, hydroxyapatite AC, HILIC-IMACDBHA probe, HA-yne probe, isoDTB tag, photo-pTyr-scaffold probe, sulfonyl-triazole probe
AcetylationLys, Ser, Thr, N terminusimmunoenrichment, SCX, ZIC-HILIC, COFRADIC, antibody-IEF, antibody-SCXalkyne-containing thioester probe, metabolic labelling by ethyl fluoroacetate
MethylationLys, Arg, His, Ala, Asnimmunoenrichment, IEF, SCX, HILIC, antibody-SCX, antibody-HpH RP, 3xMBT methyl-binding domains, antibody-propionylationazide- and alkyne-analogues of SAM, chemoenzymatic labelling by ProSeAM
GlycosylationAsn, Arg (N-linked) Ser, Thr, Tyr (O-linked)lectins, modified glycosidases, immunoenrichment, IMAC, MOAC, HILICmetabolic labelling by: Ac4ManNAz, Ac4GalNAz, Ac4FucAl, Ac4ManNAl, GalNAz, 1,3-Pr2GalNAz, GalNAzMe; chemoenzymatic labelling by UDP-GalNAz, Glyco-TQ
UbiquitinationLys, N terminus, Cys, Ser, Thrprotein degradation, trafficking, regulation of enzymes, translation, DNA repairtagged-Ub, immunoenrichment, COFRADIC
SumoylationLysprotein interactions, subcellular localization, enzymatic activitiestagged-SUMO, immunoenrichment, SUMO-interacting motifs

IMAC: Immobilized Metal Ion Affinity Chromatography

MOAC: Metal Oxide Affinity Chromatography

HILIC: Hydrophilic Interaction Liquid Chromatography

SCX/SAX: Strong Cation/Anion Exchange

COFRADIC: Combined Fractional Diagonal Chromatography

ERLIC: Electrostatic Repulsion Hydrophilic Interaction Chromatography

Learn about other Q&A about other technologies.

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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:

StepDetails
Sample TypeRecombinant TcPolβ phosphorylated in vitro by 4 kinases (TcCK1, TcCK2, TcAUK1, TcPKC1)
Sample PrepIn-gel digestion of TcPolβ bands from SDS-PAGE
EnrichmentPhosphopeptide recovery from trypsin-digested gel bands
PlatformNanoLC-MS/MS on high-resolution instruments
BioinformaticsMaxQuant-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.

KinasePhosphorylated Residues on TcPolβ
AUK1S 69S 275T 366
CK2S 69S 275T 382
CK1S 13S 46T 49S 69
S 185S 193S 275T 278
Y 307S 336T 338T 345
T 382


PKC1S 13S 46T 49S 69
S 185S 275T 382Y 302

Unexpected Dual-Specificity:

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

Publications

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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

Proteomics Sample Submission Guidelines

Ensure your samples are prepared and submitted correctly by downloading our comprehensive Proteomics Sample Submission Guidelines. This document provides detailed instructions and essential information to facilitate a smooth submission process. Click the link below to access the PDF and ensure your submission meets all necessary criteria.

Proteomics Sample Submission Guidelines
* For Research Use Only. Not for use in diagnostic procedures.
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