How to use LC-MS/MS for high-throughput peptidomics analysis?
Peptidomics is the study of endogenous peptides naturally present in biological samples.These endogenous peptide fragments(usually less than 10 kDa) are involved in physiological activities such as neural regulation, immune response, hormone signaling, and metabolic control, possessing significant functional and potential biomarker value. Compared to proteomics, peptidomics is closer to the actual cleavage, processing, and post-translational modifications of proteins.It reflects the dynamic regulation of biological systems more sensitively.However, due to the short, low-abundance, and highly isomeric nature of peptides, research in this field is quite challenging.
To overcome these challenges, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the most critical technology platform in high-throughput peptidomics analysis. It not only accurately identifies peptide sequences but also allows for high-throughput, wide-coverage, and multi-sample parallel expression difference analysis.
1. The Role of LC-MS/MS in High-Throughput Peptidomics Analysis
1. What is LC-MS/MS?
（1）LC（Liquid Chromatography）It separates complex peptide mixtures, introducing them into the mass spectrometer in order of hydrophilicity/hydrophobicity, thereby improving identification rates.
（2）MS（Mass Spectrometry）Measures the mass (m/z) of peptides, providing a preliminary estimate of their molecular weight.
（3）MS/MS（Tandem MS）Fragments target peptides and detects their fragmentation patterns again to deduce the amino acid sequence.
In peptidomics, LC-MS/MS technology enables the separation, identification, and quantification of endogenous peptides from complex biological samples.The entire process of separation, identification, and quantificationof endogenous peptides.
2. Detailed Process Analysis of High-Throughput Peptidomics
1. Sample Collection and Peptide Enrichment
Endogenous peptides are widely present in the following samples:
(1) Serum/Plasma
(2) Urine
(3) Cerebrospinal Fluid
(4) Tissue Homogenates
(5) Cell Supernatants or Lysates
Enrichment Methods:
| Method | Function | Applicable Scenarios |
| Molecular weight cutoff (<10kDa) | Removes interference from large proteins | General extraction |
| Solid-phase extraction (C18, C8) | Retains hydrophobic small peptides | Low concentration samples like urine and cerebrospinal fluid |
| Magnetic bead enrichment | Targeted cleaning and concentration | Trace samples, low-abundance analysis |
| HILIC pre-treatment | Increases the identification rate of polar peptides | Polar peptide mining |
Biotage uses a multi-strategy combination enrichment scheme to enhance the capture efficiency of short peptides and modified peptides.
2. Liquid Chromatography Separation (LC)
Endogenous peptides are diverse and have complex retention behaviors. The primary task in the LC stage isto separate peptides according to their physicochemical properties before entering the mass spectrometer,improving identification depth.
(1) Usenano-flow nanoLC systems(such as EASY-nLC 1200) to improve sensitivity.
(2) Commonly usedC18 reverse-phase chromatography columnswith a water-acetonitrile gradient elution buffer system.
(3) The separation time is usually 60–120 minutes to balance throughput and depth.
3. Tandem Mass Spectrometry Acquisition (MS/MS)
Common Mass Spectrometry Acquisition Modes:
| Mode | Characteristics | Applicable Scenarios |
| DDA (Data Dependent Acquisition) | Fragmentation by the Most Intense Peaks | Discovery-Based Research |
| DIA (Data Independent Acquisition) | Comprehensive Window Acquisition | High Quantitative Stability, Suitable for Large-Scale Screening |
| PRM (Parallel Reaction Monitoring) | Targeted Quantification of Specific Peptides | Validation Experiments or Biomarker Verification |
Biotech Pack supports DDA+DIA combined acquisition, integrating peptide database-assisted identification to enhance peptide resolution efficiency.
4. Data Processing and Peptide Identification
(1) Database Search
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Use tools like PEAKS, MaxQuant, Spectronaut for peptide matching
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Can access databases such as UniProt, Swiss-Prot, RefSeq
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Support customized searches for non-typical starting peptides, modified peptides, spliced peptides
(2) Quantitative Analysis
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Quantification methods like Label-free, iTRAQ, TMT are available
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Differential peptide screening: fold change + P-value + FDR (False Discovery Rate) control
(3) Functional Annotation
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GO enrichment, KEGG pathway mapping, protein-peptide reverse tracking
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Can construct a 'Peptide-Parent Protein-Functional Pathway' three-tier network
3. Technical Advantages of LC-MS/MS in High-Throughput Peptidomics
| Dimension | Advantage Description |
| Sensitivity | Nanomolar detection limit, suitable for capturing low-abundance short peptides |
| Precision | High-resolution mass spectrometry for accurate peptide sequence and modification site identification |
| High Throughput | Thousands of peptides can be identified in a single experiment |
| Broad Coverage | Simultaneous analysis of multiple modification states and source proteins |
| Expandable | Integration with proteomics and metabolomics data to build multi-omics models |
4. Research Applications of LC-MS/MS Peptidomics
Neuroscience: Analysis of neurotransmitter peptides and memory-regulating peptides (e.g., Neuropeptide Y)
Cancer Research: Identification of tumor-associated cleavage peptides, neoantigens, or microenvironment signaling peptides
Immunology: Integration with HLA peptidomics to elucidate immune recognition mechanisms
Metabolic Disorders: Discovery of endocrine regulatory peptides and their association with metabolic pathways
Pharmacodynamics Research: Monitoring dynamic changes in peptide expression pre- and post-drug intervention
5. Biotech Pack Biotechnology: Comprehensive High-Throughput Peptidomics Analysis Service
As a leading multi-omics platform service provider in China, Biotech Pack offersservices from samples to conclusionsOne-stop LC-MS/MS Proteomics Research Services, including:
✅ Handling of various sample types (tissues, liquids, cells, etc.)
✅ Enrichment strategy optimization and customized magnetic bead solutions
✅ High-resolution LC-MS/MS platforms (Orbitrap Exploris, Q Exactive, etc.)
✅ Choice of DDA, DIA, PRM, and other acquisition modes
✅ Multi-dimensional differential analysis + pathway enrichment + network construction
✅ Support for combined analysis with proteomics, metabolomics, and transcriptomics
Proteomics offers a higher-dimensional window into understanding complex biological processes, and LC-MS/MS serves as the 'core weapon' for separating, identifying, and quantifying these peptides from chaos. It allows us to delve into the real processes of protein processing, functional fragment expression profiles, and pathological regulation mechanisms.Biotech PackardWe will continue to rely on advanced mass spectrometry platforms, an experienced bioinformatics team, and a rigorous service system to help researchers maximize the scientific value of high-throughput proteomics analysis.
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