Current Status of Body Fluid Proteomics Research

Proteins are the main bearers of biological function, and research exploring biomarkers through body fluid proteomics, represented by plasma, has emerged endlessly. Low-abundance proteins are an important source of potential biomarkers, but due to the complexity of body fluids such as plasma and the limitations of detection methods, the detection of low-abundance proteins is limited, and new clinical biomarker discoveries are becoming fewer and fewer [1], while FDA-approved protein biomarkers are also declining year by year. Therefore, improving the detection level of low-abundance proteins is crucial for body fluid proteomics research.

Plasma proteomics-related research has successively topped leading journals such as CNS

Different technologies have their own advantages and disadvantages [2]. Which technology is the first choice for future body fluid proteomics? Although mass spectrometry is suitable for the discovery of biomarkers in body fluids such as plasma and mechanistic research, its application is limited by insufficient throughput and the number of proteins detected.

Characteristics of body fluid proteomics technologies represented by plasma

A Revolutionary Technology for Body Fluid Proteomics Research

In order to break through the technical limitations of mass spectrometry-based body fluid proteomics research, ProteinT, in collaboration with Tianjin University, after 6 years of dedicated research and development, testing dozens of nanomaterials, and validating over 20,000 samples, successfully developed a core product with independent intellectual property rights — n-LAPE/MS™, a liquid biopsy novel biomarker discovery platform based on next-generation nano-scale low-abundance protein enrichment mass spectrometry analysis technology, which can process 96 samples in 5 h. Combined with the latest mass spectrometry, within a 30-min effective gradient per single sample, up to 8,000+ proteins can be detected, with stable results, empowering the clinical application of body fluid proteomics.

ProteinT — n-LAPE/MS™ Platform

Exclusive Patented Nanomaterials — Low Cost, High Efficiency

The n-LAPE/MS™ kit for proteomic detection of blood and other body fluids exploits the differences in affinity of nano-magnetic beads for proteins of different abundances, preferentially adsorbing low-abundance proteins, thereby improving the ability of mass spectrometry to detect low-abundance proteins and thus increasing the number of proteins detected.

Technical principle of the n-LAPE/MS™ platform

Substantially Increased Number of Proteins Detected

Compared with the conventional method of removing high-abundance proteins, n-LAPE/MS™ increases the number of detected proteins by 2-3 times; within a 30-min effective gradient, the number of proteins detected by mass spectrometry reaches up to 8,000+, covering more than 80% of body fluid proteins, greatly increasing the probability of discovering new high-performance biomarkers.

Comparison of mass spectrometry detection results for plasma proteins using different pre-processing methods

Deep Measurement

After enrichment, the effectively detected protein content distribution spans more than 10 orders of magnitude; the proportion of low-abundance proteins increases from 2.54% to 54.41%, and the number of low-abundance proteins below 1 ng/mL increases from 40+ to 1,100+, with a significant increase in the number detected and more complete protein detection [3].

Stable Detection Data, High Reproducibility

The enrichment kit offers stable performance; through mass spectrometry detection, the median CV value of technical replicate protein expression is <10%, comparable to the CV value of the mass spectrometer, indicating that the pre-processing workflow is stable and does not significantly increase the dispersion of mass spectrometry results. Stable detection can lay a solid foundation for large-cohort plasma proteomics research and the discovery of high-quality biomarkers.

Automated Sample Pre-processing

Automation can truly address the throughput demands of large-cohort samples and reduce human error. ProteinT has integrated resources to develop a supporting automated pre-processing platform, PT-ASP480 Pro™, which can complete pre-processing of 96 samples in 5 h, simplifying experimental operations, reducing human factors, and lowering batch effects.

PT-ASP480 pro™ automated pre-processing platform

Small Sample Volume and Diverse Sample Types

Using this method for plasma proteomics research requires only 10 uL of plasma, conserving precious samples. At the same time, the magnetic beads have been optimized to develop kits adapted to different sample types, expanding the variety of body fluids and increasing the possibility of body fluid biomarker discovery.

Number of proteins detected within a 30-min effective gradient based on the HFX mass spectrometry platform

Sample Submission Recommendations

Sample quality is crucial to test results. In addition to common factors affecting protein detection such as hemolysis and repeated freeze-thaw cycles, the time left at room temperature also has a huge impact on detection results. Therefore, taking plasma samples as an example, according to the national standard for plasma sample collection [4], the collection standards are as follows:

1. Collect fresh venous blood using the /EDTA blood collection tube provided by ProteinT

2. Within 2 h, centrifuge at 1,300 g for 10 min and collect the upper-middle plasma layer

3. Freeze and store the plasma at -80 ℃ (can be temporarily stored at -20 ℃ for a short time), and ship on dry ice

Impact of sample collection methods on detection results

ProteinT provides high-quality body fluid proteomics services based on n-LAPE/MS™, achieving a revolutionary breakthrough in the number of proteins detected in body fluids represented by plasma and empowering the clinical translation of body fluid proteins. Meanwhile, the n-LAPE/MS™ body fluid protein pre-processing kit will soon be fully available for sale, including a complete set of reagents for low-abundance protein enrichment and protein and polypeptide extraction, shortening the manual protein pre-processing procedure to 3 h. Stay tuned!

References:

[1]  Geyer P E , Holdt L M , Teupser D ,et al.Revisiting biomarker discovery by plasma proteomics[J].Molecular Systems Biology, 2017, 13(9):942.

[2] Suhre K, McCarthy MI, Schwenk JM. Genetics meets proteomics: perspectives for large population-based studies. Nat Rev Genet. 2021 Jan;22(1):19-37

[3] Ma C, Li Y, Li J, Song L, Chen L, Zhao N, Li X, Chen N, Long L, Zhao J, Hou X, Ren L, Yuan X. Comprehensive and deep profiling of the plasma proteome with protein corona on zeolite NaY. J Pharm Anal. 2023 May;13(5):503-513.

[4] GB/T 38576-2020 "Collection and Processing of Human Blood Samples".