Whole Proteome profiling (Shotgun Protein Identification)

What is Shotgun proteomics

Proteins, which are encoded by the genome, serve as key biological actors within cells and drive many integral biological processes in both healthy and diseased states. Shotgun proteomics, also known as "Bottom-up proteomics", is a widely used and mature technology for protein identification and characterization of their amino acid sequences along with post-translational modifications (PTMs). This technique requires the proteolytic digestion of proteins prior to mass spectrometry analysis. Shotgun proteomics has been demonstrated to be a valuable tool for the identification of novel large or small proteomes and protein complexes, enabling the discovery of previously unknown protein-protein interactions. The most distinctive feature of shotgun proteomics is that it enables identify a wide range of proteins at the same time with minimal protein separation needed.

In a typical shotgun proteomics experiment, a mixture of intact proteins is digested with one or more proteases and the resulting peptides are separated in HPLC coupled with MS/MS, and followed by MS raw data analysis. Peptide identification is accomplished by comparing the tandem mass spectra obtained from peptide fragmentation with theoretical tandem mass spectra generated through in silico digestion of a protein database. Protein ID is accomplished by assigning identified unique peptide sequences to proteins.

Figure 1. A general pipeline for the identification of proteins in shotgun proteomics ^[1].

Features of shotgun proteomics

• Identification of proteins on a large scale or global proteome level;
• Quantitative comparisons analysis between samples;
• Ideal for discovery study of protein biomarker;
• Systematically profile dynamic proteomes;
• Post-translational modification discovery.

Our shotgun protein identification service

The high-throughput shotgun proteomics approach provided by Creative Proteomics are powerful tools that facilitate the identification and quantification of thousands, or even millions of proteins from a single analysis for large-scale protein profiling. The proteome of various samples can be thoroughly investigated by our team, including expression profiling, subcellular localization analysis, protein-protein interactions assessment, PTMs characterization and turnover dynamics monitoring across different temporal and spatial scales as well as cell types.

Technological Advantages

1) Extraction and separation proteins from both eukaryotic and prokaryotic organism samples, such as sub-organelle, tissues, cells, body fluids, etc.;

2) Interested proteins or peptides enrichment methods, such as IP-MS/MS;

3) Targeted proteomics techniques, such as PRM, SRM/MRM;

4) Different MS/MS detection mode, DDA vs DIA;

5) High-performance liquid chromatography (HPLC) instrumentation and High resolution mass spectrometer, such as Triple TOF 5600, Q-Exactive, Q-Exactive HF, Orbitrap Fusion™ Tribrid™.

Results Delivery

1. Detailed report, including experiment procedures, parameters, etc.

2. Raw data and data analysis results

How to place an order

At Creative Proteomics, experienced proteomics research team, strict quality control system, together with ultra-high resolution detection system and professional data pre-processing and analysis capability, ensure reliable and accurate data. Our team of experienced experts will provide a feasible experimental scheme tailored to your specific requirements, ensuring high-quality results for protein analysis. Please do not hesitate to reach out to us via email for a detailed discussion regarding your specific requirements. Our team of customer service representatives is available 24 hours a day, from Monday to Sunday.

Reference

1. Dupree EJ, Jayathirtha M, Yorkey H, et al. A Critical Review of Bottom-Up Proteomics: The Good, the Bad, and the Future of this Field. Proteomes. 2020 Jul 6;8(3):14.

Quantitative Shotgun Proteome Analysis by Direct Infusion

Journal: Nature Methods

Published: 2020

Main Technology: Unbiased quantitative proteomics, data-independent acquisition mass spectrometry (DIA-MS).

Highlights

1. We discovered that gas-phase separation can substitute for liquid chromatography (LC) to deliver expeditious analysis of complex peptide mixtures from the human proteome.
2. Using Direct Infusion – Shotgun Proteome Analysis (DI-SPA) by DIA-MS, we demonstrate the targeted quantification of over 500 proteins within minutes of MS data collection (~3.5 proteins/second).
3. We show the utility of this technology to perform a complex multifactorial proteome study of interactions between nutrients, genotype, and mitochondrial toxins in a collection of cultured human cells. More than 45,000 quantitative protein measurements from 132 samples were achieved in only 4.4 hours of MS data collection.

In conclusion, enabling fast, unbiased proteome quantification without LC, DI-SPA offers an approach to boosting throughput critical to drug and biomarker discovery studies that require analysis of thousands of proteomes.

Background

Shotgun proteomics methods using LC-MS achieve the greatest depth and breadth of proteome coverage. LC-MS delivers sensitive peptide analysis for proteomics, but the methodology requires extensive analysis time, hampering throughput. Based on theoretical results of computational analysis of how effectively gas-phase fractionation by High-field asymmetric waveform ion mobility spectrometry (FAIMS) and a quadrupole mass filter purify peptide cations, reveals that gas-phase fractionation can theoretically reduce the complexity of peptide ions for analysis without LC.

Figure 1. Overview of DI-SPA by DIA-MS strategy for peptide identification.

Q: Why is Proteome Profiling conducted?

The term "Proteome" refers to all the proteins expressed by a species, cell, or tissue. The purpose of Proteome Profiling is to identify the components of as many peptides and protein mixtures in a sample as possible. Whole proteome profiling based on mass spectrometry technologies can provide reference information for high-throughput protein quantification and modification analysis.

Creative Proteomics conducts Proteome Profiling using high-precision liquid chromatography-mass spectrometry (LC-MS) technology. This approach enables the analysis of protein types in complex samples and includes related bioinformatics analyses, such as protein identification, GO classification, and metabolic pathway analysis, providing powerful tools for proteomics.

Q: Which species can currently undergo Proteome Profiling?

Any species with sufficient data in proteomic databases, known genomes, or rich EST (or transcriptome) data can undergo Proteome Profiling. For species that do not meet these conditions, it is possible to perform genome or transcriptome sequencing alongside Proteome Profiling to support the analysis. Therefore, theoretically, all species can undergo Proteome Profiling.

Q: What are the advantages and disadvantages of different types of whole proteome profiling?

SDS-PAGE-LC-MS/MS analysis separates proteins in the sample using electrophoresis, providing good separation and high resolution for proteins with molecular weights between 10 kDa and 100 kDa. However, it faces challenges in separating proteins with molecular weights below 10 kDa, above 100 kDa, low-abundance proteins, extremely acidic or basic proteins, and hydrophobic proteins (such as membrane proteins).

HPLC-LC-MS/TS analysis separates proteins in the sample using high-performance liquid chromatography (HPLC), offering high separation efficiency, sensitivity, a wide application range, and fast analysis speed. This separation method is particularly effective for separating acidic or hydrophobic proteins. The drawback of HPLC is the presence of the "column outside effect," where any diffusion and retention of separated substances significantly broaden chromatographic peaks, leading to false positives.

Q: How should one choose a whole proteome profiling method?

When the analyzed objects include hydrophobic proteins with strong polarity, large molecular weights (>100 kDa) or small molecular weights (<10 kDa), it is recommended to use the HPLC-LC-MS/MS analysis method. When the analyzed objects are not primarily hydrophobic proteins with strong polarity and the molecular weights are concentrated in the 10-100 kDa range, it is advisable to use the SDS-PAGE-LC-MS/MS analysis method.

Q: How many protein species can generally be identified through whole proteome profiling?

This depends on the complexity of the sample being analyzed, the completeness of the database, protein content, and the degree of protein separation in the proteome. Typically, whole proteome profiling can identify 1000-3000 protein species. For example, samples derived from plants usually contain more protein species and quantities than samples from animals, and animal tissue samples typically have more protein species and quantities compared to blood samples.