Applying Empore™ E3technology™ to Proteomic Sample Preparation

E3technology™ delivers equivalent or better performance than established methods (FASP, SP4, SPEED, S-tip) for proteome identification and quantitation, with high reproducibility (Pearson > 0.95) across multiple sample types and formats.

Authors: Gregory Davison, Xiaohui Zhang, Guotao Lu, Cheng Tse Huang

Introduction

Proteomic sample preparation remains one of the most critical — and often most frustrating — steps in mass spectrometry-based proteomics. Traditional methods like FASP, SP4, and SPEED each have limitations in terms of throughput, reproducibility, and ease of use.

CDS Empore™ E3technology™ offers a new approach to protein cleanup and digestion that addresses these challenges. Available in multiple formats — E3tips (10 µL and 200 µL), E3filter (0.5 mL), and E3plate (1.2 mL) — E3technology™ is designed to handle a wide range of sample types, from bacterial cells to mammalian tissues to human body fluids.

This application note evaluates different formats of E3technology™ and tests a variety of sample types including E. coli, yeast, mammalian cells, mouse tissues, and human body fluid. The technology has shown great advantages over existing methods in the context of proteome-wide identification and quantitation.

Experiment Setup

Materials and Solutions

• Cell lysis: SDS buffer (4% SDS, 100 mM Tris-HCl, pH 8.0), Trifluoroacetic acid (TFA), TCEP (1.0 M stock), Chloroacetamide/CAA (1.0 M stock)

• Protein digestion: Rinse/precipitation/wash (RPW) buffer (80% acetonitrile), Digestion buffer (50 mM TEAB, from 1.0 M stock)

• Peptide elution: Elution I (0.2% formic acid in water), Elution II (0.2% formic acid and 50% ACN in water)

General Experimental Procedure

1. Cell lysate — Collect cell pellets (E. coli, HEK293, yeast, etc.) and rinse two times with PBS. Generate lysate by adding 100–200 µL of SDS buffer, and boil with 10 mM TCEP/40 mM CAA at 95°C for 10 min. For TFA lysate, add 50–100 µL of pure TFA to cover cell pellets and incubate at room temperature for 3–5 min.

2. Protein precipitation and cleanup — Add 4× volume of RPW buffer, transfer protein precipitates to E3tip, E3filter, or E3plate. Spin to discard flow through. Wash 2–3 times with RPW buffer and discard flow through.

3. Protein digestion and peptide elution — Add 100–200 µL of digestion buffer and an optimized ratio of proteolytic enzyme. Incubate filters at 37°C for 4–15 hours with gentle shaking. To elute peptides, add sequentially Elution I and II, spin, and collect. Dry peptides in SpeedVac.

4. Peptide desalting — Refer to Application Notes #278, #259, and #272 for detailed protocols using Empore™ C18 Spin Columns and StageTips.

5. Proteome identification and quantification — LC-MS/MS performed using an Ultimate 3000 RSLCnano system coupled with an Eclipse Orbitrap mass spectrometer. Linear gradient of 110 min from 1% to 35% buffer B (0.1% formic acid in acetonitrile). Spectra acquired in data-dependent mode. Proteome Discoverer (v2.5) and MaxQuant used for protein database search.

Critical note: The conditioning step is essential — E3technology™ membranes are hydrophobic and must be pre-wetted with a water-miscible solvent (methanol) for uniform flow and good recovery. If the membrane dries accidentally, repeat the pre-wetting step.

Results and Discussion

E3technology™ was benchmarked against several established methods — FASP, SP4-GB, SPEED, and S-tip — using E. coli as a model system.

Key findings from the E. coli benchmarking study:

• Equivalent or better protein identification compared to FASP and SP4-GB methods

• High reproducibility with Pearson correlation > 0.95 between replicates

• Lower coefficient of variation for both protein and peptide quantification compared to FASP and SP4-GB

• Fewer missed cleavages indicating more complete protein digestion

E3technology™ was also validated across diverse biological samples — HeLa cells, mouse kidney tissue, and human saliva — demonstrating its versatility for different sample types and complexities.

E3technology™ was also validated across diverse biological samples — HeLa cells, mouse kidney tissue, and human saliva — demonstrating its versatility for different sample types and complexities.

Key takeaway: E3technology™ provided equivalent or better performance in terms of proteome identification and quantitation across all tested sample types. The reproducibility of the technology is generally high (Pearson > 0.95), and it can be easily scaled and automated using the plate format.

Conclusion

E3technology™ provides equivalent or better performance than established proteomics sample preparation methods including FASP, SP4, SPEED, and S-tip. The technology is rapid, reliable, reproducible, easy to handle, and stress-free. With the E3plate format, it can be easily scaled and automated for high-throughput applications.

E3technology™ significantly lowers the technical and economic barriers to proteomics experiments, offering a powerful and versatile platform for proteome science.