ProtiFi S-Trap Midi Spin Columns for Proteomics Sample Preparation

ProtiFi S-Trap™ Midi Spin Columns are centrifugal columns for protein sample preparation prior to LC-MS/MS analysis. The Midi format processes up to 1 mg of total protein and uses patented S-Trap™ technology to combine SDS-based protein solubilization, reduction, alkylation, trapping, contaminant removal, and subsequent enzymatic digestion directly within the column.

S-Trap™ Technology

Protein samples are first solubilized and denatured in an SDS-containing solution. Reduction, alkylation, and acidification to pH ≤1 are followed by addition of a Binding/Wash Solution containing 90% methanol. Under these conditions, proteins are trapped within the S-Trap™ matrix. 

The column is washed to remove components incompatible with downstream mass spectrometry. Protease is then added directly to the matrix for enzymatic digestion. The resulting peptides are sequentially eluted and can be concentrated for subsequent LC-MS/MS analysis. 

Standard Workflow

1. Protein solubilization and denaturation
2. Reduction with TCEP
3. Alkylation with MMTS
4. Acidification to pH ≤1
5. Addition of Binding/Wash Solution
6. Protein trapping in the S-Trap™ Midi Spin Column
7. Washing and contaminant removal
8. Addition of the selected protease
9. Digestion for 1–2 hours at 47 °C
10. Sequential peptide elution
11. Lyophilization or SpeedVac concentration and preparation for LC-MS/MS

Technical Specifications

• Format: Centrifugal spin column
• Application: Proteomics sample preparation
• Maximum Total Protein Amount: ≤1 mg
• Protease: application dependent; e.g., trypsin or Trypsin/Lys-C
• Protease-to-Protein Ratio: 1:10 to 1:100 (w/w)
• Standard Protein-Trapping Centrifugation: 4,000 × g for 30 s
• Maximum Centrifugation if Flow Is Insufficient: up to 8,000 × g for 5 min
• Washing: 3.0 mL Binding/Wash Solution, 3 times
• Digestion Solution Volume: 350 µL
• Digestion: 1–2 h at 47 °C
• First Elution: 500 µL Digestion Solution
• Second Elution: 500 µL Elution Solution
• Optional Third Elution: 500 µL Elution Solution 2 for hydrophobic peptides
• Flow Methods: centrifugation; optional vacuum or positive pressure
• Downstream Analysis: LC-MS / LC-MS/MS
• Intended Use: Research Use Only – not for use in diagnostic procedures

S-Trap™ Workflow Reagents

The current protocol specifies:

• Solution 1 – 2× Lysis Solution: 10% SDS, 100 mM TEAB, pH 7.55
• Solution 2 – Reductant: 120 mM TCEP
• Solution 3 – Alkylator: 500 mM MMTS in IPA
• Solution 4 – Acidifier: 12% phosphoric acid
• Solution 5 – Binding/Wash Solution: 100 mM TEAB in 90% methanol, pH 7.5
• Solution 6 – Digestion Solution: 50 mM TEAB, pH 8.0
• Solution 7 – Elution Solution: 0.2% formic acid in water
• Solution 8 – Elution Solution 2: 50% ACN in 0.2% formic acid, optional for hydrophobic peptides. 

Reagents and protease are not included with the S-Trap™ Midi Spin Columns themselves. ProtiFi recommends LC-MS-grade reagents where applicable.

Available Pack Sizes

• PN C002-MIDIX-0010PK – 10 S-Trap™ Midi Spin Columns
• PN C002-MIDIX-0020PK – 20 S-Trap™ Midi Spin Columns
• PN C002-MIDIX-0040PK – 40 S-Trap™ Midi Spin Columns