A complete guide to the workflow of subcellular proteomics (including mass spectrometry and...)

A complete guide to the workflow of subcellular proteomics (including mass spectrometry and...)SILAC Technology) Testing Technology Service Details Introduction

In the post genomic era, subcellular proteomics is gradually becoming a key research direction for revealing cellular functions, signaling pathways, and disease mechanisms. Compared to whole cell level d à nb á izh ì omics, subcellular level studies focus on protein expression, localization, and dynamic changes within specific organelles (such as mitochondria, endoplasmic reticulum, nucleus, etc.), providing higher spatial resolution data support.

1、 Why choose subcellular d à nb á izh ì omics?

Although traditional omics has achieved extensive results in screening disease biomarkers and studying signaling pathways, its spatial resolution is often limited. For example, in tumor cells, changes in protein expression at the whole proteome level do not directly reflect the phenomenon of protein translocation at the organelle level.

Subcellular d à nb á izh ì omics, through the separation and quantitative analysis of organelles, can:

• Reveal the dynamic transport of proteins between different organelles.

• Identify local protein expression abnormalities related to the disease.

• Reconstruct the protein network structure in the spatiotemporal dimension.

In this research direction, the joint application of high-resolution mass spectrometry platforms and quantitative labeling strategies is particularly crucial.

2、 The core workflow of subcellular d à nb á izh ì omics

1. Sample preparation and subcellular component separation

High quality subcellular separation is the foundation of the entire process. The current mainstream methods include:

• Differential centrifugation: suitable for rough separation of most organelles such as mitochondria, nuclei, vesicles, etc.

• Density gradient centrifugation (such as sucrase or Percoll gradient): used for further purification of target organelles.

• Immunoaffinity separation: Enriching specific organelles with antibodies, suitable for cell structures that are difficult to centrifuge.

• Subcellular Localization Mapping: Combining techniques such as SILAC to enhance spatial resolution.

2. Protein extraction and enzyme digestion

There are significant differences in the types and abundance of proteins in different organelles, so it is necessary to optimize the buffer system during protein extraction, such as:

• For samples enriched in m ó d à nb á i, it is recommended to use high salt and non-ionic detergents.

• Nucleic acid containing nuclear components require the addition of nucleases to reduce sample viscosity.

Enzymatic cleavage is usually performed using y í d à nb á im é i (Trypsin), which may be combined with coenzymes such as Lys-C when necessary to enhance the efficiency of complex protein cleavage.

3、 Quantitative Strategy Explanation: Advantages of SILAC in Subcellular D à nb á izh ì omics

1. What is SILAC?

SILAC（Stable Isotope Labeling by Amino acids in Cell culture） It is a metabolic labeling strategy that achieves relative quantification of d à nb á izh ì levels by introducing amino acids containing diazo isotopes during cell culture. Its advantages include:

• No chemical modification is required to avoid introducing non physiological changes.

• Suitable for dynamic transportation research.

• Naturally compatible with mass spectrometry, with high quantitative accuracy.

In subcellular d à nb á izh ì omics, SILAC is particularly suitable for:

• Construction of protein localization map: Different SILAC labeling groups simulate different organelles and infer localization based on label intensity and distribution patterns.

• Dynamic transport study: Changes in SILAC tag intensity before and after processing, revealing protein migration trends.

• Omics comparison under high-throughput screening conditions.

2. SILAC vs other quantitative techniques (TMT, Label free)

4、 Mass spectrometry analysis and data interpretation

1. Instrument selection: high-resolution vs high-throughput

Subcellular d à nb á izh ì omics typically recommends the use of high-resolution mass spectrometers (such as Orbitrap Exploris, timsTOF series) to enhance the detection capability of low abundance proteins and isotope labels.

2. Data processing flow

The typical analysis process includes:

• Raw to mzML data preprocessing.

• Isotope labeling identification and quantification (MaxQuant, Proteome Discoverer, etc.).

• Subcellular localization prediction software assistance (such as MetaMass, SubCons, etc.).

• Enrichment analysis and functional annotation (GO, KEGG, Reactome).

In practical projects, quality control of data is particularly crucial. For example:

• Is the protein coverage balanced.

• Is the resolution of isotope peaks sufficient.

• Is there batch effect interference.

Subcellular d à nb á izh ì omics is an important bridge connecting protein expression and cellular function. With the improvement of mass spectrometry sensitivity and diversification of quantitative techniques, subcellular level j ī ngqu è omics has become a means of understanding complex life phenomena. In the future, new directions such as spatial multi omics, AI assisted localization prediction, and single-cell subcellular omics will further expand the research boundaries. Baitai Paike Biotechnology will continue to optimize its one-stop service process from sample processing, mass spectrometry analysis, to bioinformatics interpretation, helping researchers explore the mysteries of life at higher resolutions.

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Baitai Paike Biotechnology Characteristic Project

1、 Protein sequencing

Biotec Biotech uses Thermo's newly launched Obitrap Fusion Lumos mass spectrometer and Shimadzu's Edman degradation sequencing system to analyze the d à nb á izh ì sequence, providing mass spectrometry based protein sequencing analysis services, including amino acid composition analysis of d à nb á izh ì, Ndu ā nc è x ù, Cdu ā nc è x ù, and full sequence analysis, as well as d à nb á izh ì N-terminal sequence analysis services based on Edman degradation. For unknown theoretical sequences, we provide de novo sequencing services based on de novo sequencing to analyze protein sequences.

※ Service advantages:

1. Adopting the world's advanced mass spectrometer Obitrap Fusion Lumos;

2. It can achieve 100% coverage of the measured target protein sequence;

3. It can determine up to 70 amino acid sequences at the N-terminus of proteins;

4. Can measure various forms of samples: protein solution, PVDF protein bands;

5. Low sample usage: Protein samples only require 5-10ug to complete detection;

6. Sequencing is not affected by N-terminal modifications such as blocking, PEC, and glycosylation.

2、 D à nb á izh ì omics

Biotec Biotech uses Thermo Fisher's Orbitrap Fusion Lumos mass spectrometry platform combined with Nano LC to provide a variety of d à nb á izh ì omics analysis services, including quantitative d à nb á izh ì omics, targeted d à nb á izh ì omics, proteomics, post-translational modified proteomics, and more. In addition, Baitai Paike Biotechnology has launched a 4DD à nb á izh ì omics service based on timsTOF Pro, which helps with research in micro sample proteomics, large sample population medicine, and high-throughput modification genomics.

※ Service advantages:

1 . High throughput quantitative protein analysis: Large scale experimental analysis with multiple control groups, discovering new biomarkers;

2. Multiple in vitro and in vivo labeling methods are suitable for analyzing various samples such as tissues, cells, and blood;

3. High sensitivity of mass spectrometry analysis and high repeatability of experimental results;

4. Can detect low abundance proteins with a wide linear range;

5. Bioinformatics analysis is more systematic and accurate.

3、 Single cell mass spectrometry flow cytometry analysis

Baitai Paike Biotechnology uses the Fluidigm mass spectrometry flow cytometry system for single-cell mass spectrometry analysis. Metal element markers (usually metal element labeled specific antibodies) are used to label molecules on the surface and inside of cells. Then, individual cells are separated using flow cytometry principles, and the atomic mass spectrum of individual cells is analyzed using inductively coupled plasma mass spectrometry (ICP-MS). The atomic mass spectrum data is then converted into signal molecule expression levels on the cell surface and inside.

※ Service advantages:

1. Advanced technology, filling technological gaps

The use of metal labeled antibody technology avoids the problem of few and easily interacting channels in traditional flow cytometry fluorescence. At the single-cell level, multiple indicators can be characterized simultaneously, and Baitai Paike Biotechnology can detect 51 target proteins simultaneously.

2. Large analysis quantity and low cost

Single cell RNAseq is limited by factors such as cost, and the total number of analyzed cells for all samples is generally around 2x10 ^ 4. However, flow cytometry technology can analyze at least 10 ^ 5 cells in one sample, achieving an order of magnitude increase, and the cost is not higher than that of single-cell RNAseq.

3. Great application prospects

① The results of flow cytometry can provide changes in cell subpopulations, which have great research prospects in clinical diagnosis, disease mechanism research, and other fields;

② Combining metal tag technology with other technologies will lead to new application directions. In addition to conventional proteins, mass spectrometry flow cytometry technology can also be used for post-translational modification of proteins;

③ Can detect cell survival rate, cell size, mRNA transcript expression level, DNA synthesis rate, and protease activity.

4、 Immunopeptide omics analysis and novel antigen discovery based on high-precision mass spectrometry

The one-stop solution for high-precision mass spectrometry based immunopeptidomic analysis and new antigen discovery from Biotec Biotech includes our proprietary, highly sensitive immunopeptide enrichment and identification scheme. We can assist you in identifying and recognizing over 10000 type I peptides and over 10000 type II peptides. Through our optimized high-throughput immunopeptidomic analysis platform, immunopeptidomic analysis can be performed with reproducible identification and quantification from small sample materials. This service can be applied to large-scale research, aiming to assist researchers in finding solutions for cancer, immune diseases, and infectious diseases, and to explore unknown targets in depth.

5、 Characterization of Biopharmaceuticals

Baitai Parker is based on high-resolution mass spectrometry technology, MALDI TOF， High performance chromatography separation technology provides a range of biological drug analysis solutions, from amino acid composition and primary structure analysis of biological products such as d à nb á izh ì, peptides, antibodies, vaccines, to product variability and purity analysis. Intended to provide high-quality biopharmaceutical analysis services and assist biopharmaceutical manufacturers in improving the quality of biopharmaceuticals.

Baitai Paike Biotechnology's Seven Major Testing Platforms

Baitai Paike Biotechnology - High quality service provider for characterization of biological products and multi omics of biological mass spectrometry

Beijing Baitai Paike Biotechnology Co., Ltd. is committed to providing quality control testing and project validation services for the biological/pharmaceutical and medical device industries. The company's laboratory follows regulations and guidelines such as NMPA, ICH, FDA, and EMA, and has obtained CNAS/ISO9001 dual quality system certification. It has established a complete quality system, including data cold and hot/remote backup, regular equipment measurement/periodic verification, software audit tracking, and provides customers with integrated solutions and technical services to support new drug research and development, drug application registration, and production release.

1. The company adopts the ISO9001 quality control system and provides CRO detection and analysis services based on mass spectrometry;

2. Recognized by the National CNAS Laboratory, we provide customers with drug quality research services that comply with global pharmaceutical regulations;

3. The business scope covers omics, proteomics, metabolomics, biopharmaceutical characterization, single-cell analysis, single-cell mass spectrometry flow cytometry, bioinformatics cloud analysis, and multi omics integrated analysis of biological mass spectrometry;

4. Seven major quality control testing platforms to meet your one-stop service needs;

5. Providing services to over 3000 enterprises and 10000+customers;

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A complete guide to the workflow of subcellular proteomics (including mass spectrometry and...)SILAC Technology) Testing Technology Service Details Introduction