Application of Proteonano™ Platform for Whole-Proteome Extraction

Mass spectrometry-based proteomics workflows typically include protein extraction, pretreatment, separation, and mass spectrometry. Among them, the extraction of proteins from biological samples and their enzymatic digestion into peptides is particularly critical, which determines the sensitivity and accuracy of proteomic analysis. Traditional pretreatment methods, such as liquid digestion, in-gel digestion, and filter membrane-assisted sample treatment, usually have the disadvantages of complex transfer steps and large contact areas between the sample and the vessel wall, resulting in sample loss and hindering the implementation of in-depth proteomic analysis.

The single-pot, solid-phase-enhanced sample-preparation (SP3) technology based on carboxylated hydrophilic magnetic beads has been developed to overcome the limitations of traditional methods. The SP3 method is driven by a high concentration of organic solvent to capture proteins using carboxyl groups bound to the surface of magnetic beads. Proteins bound to the SP3 magnetic beads are then digested with trypsin in aqueous solution. SP3 magnetic beads are compatible with surfactants, and digested proteins or peptides can be eluted under aqueous conditions. After elution, mass spectrometry analysis is performed, and the whole process of sample pretreatment can be completed in the same centrifuge tube.

Figure 1. SP3-based proteomic sample pretreatment

Instrument and Software

Proteonano™ proteomics platform is a highly automated and standardized proteomics sample preparation and analysis system, which consists of Proteonano™ SP3 kit and Nanomation™ G1 automated sample preparation workstation, and proteomics analysis software. The Proteonano™ kit utilizes multivalent, multi-affinity and superparamagnetic nanoprobes to enrich low abundance proteins in composite protein samples.

Proteonano™ Platform

The high-resolution LC-MS/MS instrument chosen to be used was Thermo Scientific Vanquish Neo coupled with Orbitrap Astral. Plant and animal tissue samples were homogenized by grinding with liquid nitrogen, suspended in lysis buffer, and tissue cultured cells were homogenized with cell lysis buffer.

Protein Identification of Plant and Animal Tissue Samples

The automated platform consisting of Proteonano™ SP3 Proteome Extract Kit and Nanomation™ G1 was used for protein enrichment from different tissue samples. After trypsin digestion, 300 ng of peptides were extracted through an automatic sampler. Separation was performed in the analytical column (5 μm*150 mm, C18, 2 μm, 100 A). Two mobile phases (mobile phase A: 0.1% formic acid, mobile phase B: 0.1% formic acid, 80% ACN) were used to establish an 8-min analytical gradient. The liquid phase flow rate was set to 1.8 μL/min, and the mass spectrometry results were collected in DIA mode.

Table 1: Mass spectrometry experimental parameters of different biological samples

A total of 9414 proteins were detected in frozen Arabidopsis thaliana (At.) samples treated with the Proteonano™ platform, while 9916 proteins were detected in similarly treated frozen tobacco samples. An additional 100 to 400 proteins were identified by Proteonano™ SP3 kit compared to other nanoparticle quantification detection kits. In addition, 11,934 and 12,521 proteins were detected in the fresh corn and soybean samples treated with the Proteonano™ ultra-plex platform, respectively. Compared with filter-assisted sample preparation (FASP), 800 to 1000 more soy proteins weree identified in proteins extracted by the Proteonano™ SP3 kit. More than 8300 proteins were detected from frozen rabbit intestinal organoid samples treated with the Proteonano™ platform, and the results were similar to those obtained with other commercial SP3 assays.

Figure 2. Protein identification counts for plant and animal tissue samples

Protein Identification of Hela Cells

10μg HeLa cell protein lysates were processed using the Proteonano™ ultra-plex proteomics platform and then analyzed using Orbitrap Astral coupled with Vanquish NEO liquid chromatography system. After a 13 min analytical gradient, mass spectrometry results were collected in DIA mode. The use of Proteonano™ platform enabled the detection of 7198 proteins in HeLa cell lysates. Protein analysis of samples treated with different beads using the Proteonano™ SP3 kits revealed that more than 6,800 proteomes could be detected, with the most proteins identified in samples treated with the hydrophilic and hydrophobic bead-based kits.

Figure 3. Number of proteins identified in Hela Cells Enriched by Proteonano™ SP3 Proteome Extract Kit with different hydrophobic and hydrophilic properties (Hydrophilic (HL); Hydrophobic (HP); Amphiphilic (AP))

Benchmarking the Proteonano™ Proteomics Platform

Samples prepared using the Proteonano™ proteomics platform and samples prepared using the conventional organic reagent precipitation method were identified by a Vanquish NEO liquid chromatography system in combination with an Orbitrap Astral mass spectrometer. Data was collected in DIA mode.

Table 2. MS experimental parameters for different biological samples

The analysis results of Hela cell protein samples treated by Proteonano™ high-throughput proteomics platform showed accuracy and data consistency. The concentration of peptides detected by mass spectrometry showed a linear growth relationship with different initial sample protein amounts, indicating that peptide concentrations could be detected and quantified within a certain range of sample protein amounts. In addition, the correlation coefficients for the identification results of Hela cell proteins with initial sample protein amounts of 10 μg, 20 μg, 50 μg, 100 μg, and 150 μg were all above 0.95, further indicating that Proteonano™ platform combined with mass spectrometry could provide accurate and consistent results under different sample conditions.

With the same mass spectrometry injection volume, the proteins in the samples prepared by the Proteonano™ SP3 proteomics platform could cover at least 90% of the proteins prepared using the gradient protein precipitation method. This demonstrates that extremely low amounts of samples can be treated by the use of the Proteonano™ platform based on SP3 technology for the high-number protein analysis. The correlation coefficients between the identification results of proteins prepared by the Proteonano™ proteomics platform and those prepared by the precipitation method were all above 0.90, and the average correlation coefficient between samples was 0.96, which further showed that the Proteonano™ platform had good accuracy.