Editor's note: In its article "High-Throughput, High-Coverage Blood Proteomics Analysis Solution Based on Zeno SWATH® DIA Technology", SCIEX published the latest experimental data obtained with the n-LAPE/MS™ method platform independently developed by ProteinT, based on Zeno SWATH® DIA technology. It brings a revolutionary breakthrough to high-throughput, deep-coverage plasma protein research and clears away the bottleneck that has long hindered proteomic research on blood samples. The n-LAPE/MS™ next-generation nano low-abundance protein enrichment mass spectrometry analysis platform independently developed by ProteinT is a new biomarker discovery platform for liquid biopsy. Dozens of self-synthesised and modified nanomaterials have been stockpiled, and through a high-resolution LC-MS/MS technology platform it can specifically and effectively enrich the low-abundance proteome of blood, bringing a revolutionary increase in the number of blood proteins detected by mass spectrometry. This technology platform can effectively resolve the bottlenecks encountered in plasma proteomics and can be widely applied in clinical medical research and exploration across cancer, cardiovascular and cerebrovascular disease, drug target discovery and more, leading innovative discoveries for blood proteomics in the field of liquid biopsy.

Characterisation of the blood proteome is of great significance for clinical proteomics research, especially for biomarker discovery. Blood samples, with their extremely wide range of protein abundance and high complexity, pose enormous challenges to deep coverage and high-throughput analysis of the blood proteome.

Zeno SWATH® DIA technology combines the Zeno™ trap with SWATH® DIA technology. By raising the duty cycle to >90%, the Zeno™ trap increases MS/MS sensitivity, delivering better MS2 spectral quality and reproducibility. SWATH® DIA uses variable Q1 window acquisition so that, according to ion distribution, the ion intensity distribution in each isolation window is more uniform, while fragment information is obtained for all precursor and product ions across the scan range. As a data-independent acquisition (DIA) technique, Zeno SWATH® DIA can achieve greater protein identification depth and more precise protein quantification in a shorter time. This technology will bring new improvement and transformation to high-throughput, deep-coverage plasma proteome research.

This solution applies Zeno SWATH® DIA technology to acquire proteomic data from human plasma samples processed by the n-LAPE/MS™ method. Combined with data searching using DIA-NN software, it establishes a rapid, high-throughput analysis workflow that achieves deep coverage of the plasma proteome.

Key features of the solution:

►1. By raising the duty cycle to >90%, Zeno™ Trap technology greatly improves MS2 sensitivity, thereby enhancing MS2 spectral quality and achieving better reproducibility.

►2. Zeno SWATH® DIA integrates Zeno™ trap technology with SWATH® DIA. SWATH® DIA acquisition uses variable Q1 window acquisition, making the ion intensity distribution in each isolation window more uniform while obtaining information on all precursor and product ions within the scan range. The data obtained with this technology show good reproducibility, achieve greater protein identification depth in a shorter time and more precise protein quantification.

► 3. DIA-NN software provides analytical tools for protein identification and quantification, and can perform library-based and library-free analysis of Zeno SWATH® DIA data, enabling rapid high-throughput proteomic analysis.

► 4. By integrating Zeno SWATH® DIA technology, sample preparation technology and DIA-NN software, more than 5,500 proteins can be identified from plasma samples within a 30-min gradient, with data that show high stability and good quantitative parallelism.

Experimental methods:

Sample:Two pooled human plasma samples; 40 µL plasma samples were processed by the n-LAPE/MS™ method [1], proteins were enzymatically digested, and the peptide injection amount was 400 ng.

LC method:ACQUITY UPLC M-Class liquid chromatography system (Waters). Mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in acetonitrile. Flow rate: 5 µL/min. Elution gradient: 0–30 min, 5%–45% B; 30–34 min, 45%–80% B; 34–37 min, 80% B; 37–39 min, 80%–5% B; 39–40 min, 5%.

Mass spectrometry system:ZenoTOF™ 7600 (SCIEX), acquisition mode Zeno SWATH® DIA.

Data processing:DIA-NN software (version 1.8) [2]. The database was constructed from human cell samples fractionated by high-pH reversed-phase chromatography with data acquired by Zeno DDA [3].

Experimental results:

1. Zeno SWATH® DIA data acquisition

Zeno SWATH® DIA acquisition technology was used to collect data from the two sample groups. The total ion chromatogram showed that peptides were effectively separated within the LC gradient time, and the replicated acquisitions showed good consistency and overlap in mass spectrometry signal response and chromatographic peak shape (Figure 1).

Figure 1. Total ion chromatogram of Zeno SWATH® DIA data acquisition.

2. Zeno SWATH® DIA data analysis

DIA-NN software was used for library-based data searching. The number of proteins identified in both sample groups exceeded 5,500 and the number of identified peptides exceeded 25,000 (Figure 2-A). Venn diagram analysis showed 6,231 proteins identified in both sample groups, with a protein overlap ratio of 93.% (Figure 2-B). Correlation heat map analysis showed that the correlation of replicated acquisitions of the same sample was above 0.99 (Figure 2-C). Compared with the conventional information-dependent acquisition (IDA) technique, Zeno SWATH® DIA achieved greater coverage depth of the plasma proteome, along with better stability and good quantitative parallelism.

Figure 2. Protein identification and quantification results.

A. Bar chart of identified proteins and peptides; 

B. Venn diagram analysis of proteins identified in the two sample groups;

 C. Heat map of correlation analysis

3. Comparison with the Human Plasma Proteome Project (HPPP) data set

The proteins identified in the two sample groups were compared with the HPPP database [4]. Among them, 2,525 proteins could be matched to the HPPP database (Figure 3-A). These proteins cover the common medium- and high-abundance proteins in blood, such as albumin, alpha-2-macroglobulin, complement C3, apolipoprotein B and complement factor H. More critically, the coverage also extends to many low-abundance proteins with concentrations ≤10 ng/mL, such as angiomotin-like protein 1 (AMOL1, 0.1 ng/mL), cytoskeleton-associated protein 5 (CKAP5, 0.4 ng/mL) and platelet-activating factor acetylhydrolase β (PA1B3, 0.3 ng/mL), and even to proteins of lower abundance such as the E3 ubiquitin ligase RNF213 (RN213, 3.5 pg/mL) (Figure 3-B). In addition, applying Zeno SWATH® DIA technology combined with n-LAPE/MS™ sample preparation, more than 4,200 proteins were uniquely identified in this data set. These data further enrich and expand the human plasma proteome data set, providing more new and valuable evidence and information for disease biomarker discovery and screening, disease molecular mechanisms, drug target discovery and clinical translation.

Figure 3. Comparative analysis with the HPPP data set

A. Venn diagram comparison with the HPPP data set;

B. Overall distribution of the content of identified blood proteins

The results above show that applying Zeno SWATH® DIA technology combined with plasma sample preparation technology can achieve deep coverage of the plasma proteome, with the content of identified plasma proteins spanning more than 8 orders of magnitude. In addition, the proteomic data obtained show high stability and good quantitative parallelism, demonstrating the unlimited potential of Zeno SWATH® DIA technology for high-throughput and high-depth analysis of the blood proteome.

ProteinT

ProteinT is a national high-tech enterprise focused on the frontiers of multi-omics in precision medicine. As the leading unit of the Tianjin Key Laboratory of Clinical Multi-Omics, it conducts multi-omics research spanning genes, proteins and metabolites, and has developed advanced liquid biopsy technology based on protein mass spectrometry with independent intellectual property rights. Its core team comes from Yale University, the National Institutes of Health, the National Center for Protein Sciences and other renowned research institutions in China and abroad. The company's R&D pipeline covers early screening and early diagnosis, recurrence monitoring and companion diagnostic services for common cancer types and pan-cancer types — liver, lung, gastric, oesophageal, colorectal and pancreatic cancer — as well as cardiovascular and cerebrovascular diseases. With its Umbrella™ clinical molecular database as core intellectual property and more than 100,000 clinical sample data points, ProteinT is committed to continuous innovation in medical technology together with its commercialisation and clinical application, advancing medicine and exploring more possibilities for human health.

SCIEX

SCIEX is committed to applying innovative and precise scientific thinking, integrating reliable solutions, advancing human scientific understanding, and improving people's health and safety. We bring 50 years of innovation experience in mass spectrometry. Since the launch of the first commercial triple quadrupole mass spectrometry system in 1981, we have been dedicated to developing breakthrough technologies and solutions that influence and advance the scientific research and outcomes that improve lives. Today, as part of Danaher, a global innovator in life sciences and technology, SCIEX continues to develop robust solutions in mass spectrometry and capillary electrophoresis. We help customers monitor environmental hazards and respond rapidly; better understand diseases and disease markers to improve clinical treatment and support the development and launch of related drugs; and ensure that food is healthier and safer. This is why scientists around the world choose SCIEX products: we help you obtain reliable results so that you can make better critical decisions and improve people's lives.

References ✦

[1] Ma Congcong et al. A method for improving mass spectrometry identification data of proteins and/or peptide groups. Application No.: 202111243949.

[2] Demichev V, Messner CB, Vernardis SI, Lilley KS, Ralser M. DIA-NN: neural networks and interference correction enable deep proteome coverage in high throughput. Nature Methods. 2019. 17, 41-44.

[3] Large-scale protein identification using microflow chromatography on the ZenoTOF 7600 system. SCIEX community post, RUO-MKT-18-14415-A.

[4] Deutsch EW, Omenn GS, Sun Z, Maes M, Pernemalm M, Palaniappan KK, Letunica N, Vandenbrouck Y, Brun V, Tao SC, Yu X, Geyer PE, Ignjatovic V, Moritz RL, Schwenk JM, Advances and Utility of the Human Plasma Proteome, J Proteome Res. 2021 Dec 3;20(12):5241-5263.