How to Desalt Peptides for LC-MS/MS Analysis Using C18 StageTips: A Step-by-Step Protocol
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- 6 min read
A complete, practical guide to peptide desalting with Empore™ C18 StageTips — from sample acidification through elution — with troubleshooting tips and best practices for maximizing peptide recovery.
Authors: CDS Empore™ Technical Team
Introduction
Peptide desalting is one of the most critical — and most underappreciated — steps in any bottom-up proteomics workflow. After enzymatic digestion, your peptide sample typically contains salts, buffers, urea, detergents, or other reagents that are incompatible with LC–MS/MS analysis. These contaminants suppress ionization, reduce sensitivity, degrade spectral quality, and over time cause physical damage to rotary valves and trapping columns.
C18 reversed-phase solid-phase extraction using StageTips is the most widely used method for peptide desalting in proteomics. The principle is straightforward: peptides bind to the C18 membrane under acidic, aqueous conditions; salts and hydrophilic contaminants wash through; and clean peptides are eluted with an organic solvent. The entire process takes about 15–20 minutes and requires only a benchtop centrifuge.
This protocol uses Empore™ C18-HD StageTips (Part No. 70-2019-1001-3), which feature a high-density, 2-layer C18 membrane in a 200 µL pipette tip format. The same general principles apply to other reversed-phase StageTips (C8, SDB-XC) with minor differences in selectivity.
Who is this protocol for? Any researcher performing bottom-up proteomics who needs to clean up peptide samples before LC–MS/MS, MALDI-MS, or other mass spectrometry analysis. Suitable for in-solution digests, in-gel digests, FASP eluates, S-Trap eluates, and SP3 eluates.
Materials and Reagents
Equipment
Empore™ C18-HD StageTips, 200 µL (Part No. 70-2019-1001-3)
Benchtop centrifuge
StageTip adapters for 1.5/2.0 mL tubes (Part No. 70-2019-102X-X)
1.5 or 2.0 mL collection tubes (low-binding or low-retention recommended)
Standard pipettes (10–200 µL range)
Vacuum concentrator (SpeedVac) — optional, for final concentration
Solutions (prepare fresh)
Important: Use LC-MS grade solvents for all solutions. Contaminated solvents are a common source of polymer peaks and background noise in mass spectrometry data.
Sample Preparation
Before loading onto the StageTip, your peptide sample must be in an acidic, aqueous solution. This ensures that peptides are fully protonated and will bind efficiently to the C18 membrane.
Add 2% TFA solution to your peptide sample at a ratio of 1 part TFA solution to 3 parts sample. The final TFA concentration should be ≥ 0.5%. For example: to a 150 µL digest, add 50 µL of 2% TFA. Total sample volume should be ≤ 200 µL per StageTip.
Note: TFA is recommended due to exceptional ability as an ion pairing agent for peptides when performing reverse phase cleanup. TFA is not carried through the whole procedure as it can cause ion suppression and contamination to electrospray ionization source coupled mass spectrometers. Acetic acid and formic acid can be good alternatives to TFA as an ion pairing agent without the MS ion suppression or contamination issues.
Tip: Check pH with a small aliquot on pH paper. The pH should be ≤ 3 for efficient C18 binding.
If your sample contains precipitate after acidification (common with SDC-containing buffers), centrifuge at 14,000 × g for 5 minutes and transfer the clear supernatant to a new tube before loading.
Detergents should be used with caution as they can interfere with peptide retention. If they must be used either removed (e.g. buffer exchange) or destroyed (e.g. acid liable detergents) prior to sample loading.
Step-by-Step Protocol
All centrifugation steps are recommended at 1,000–1,500 × g, but can be modified if desired by the user. The workflow follows five stages: condition → equilibrate → load → wash → elute.
Step 1: Conditioning
Place the StageTip into a collection tube with an adapter. Add 50–100 µL methanol into the tip to wet the C18 membrane. Centrifuge at 1,000–1,500 × g until nearly all solution has passed through. Do not spin to dryness. Discard the flow-through.
If the membrane has gone dry, repeat this step. A dry membrane will have significantly reduced binding capacity.
Step 2: Equilibration
Add 50–100 µL of 0.5% TFA to the tip. Centrifuge at 1,000–1,500 × g until almost all solution has passed through. Do not spin to dryness. Discard the flow-through.
If the membrane has gone dry, repeat Steps 1 & 2 in order. A dry membrane will have significantly reduced binding capacity.
Step 3: Sample Loading
Place the StageTip into a new collection tube (low retention tube preferable). Load your acidified peptide sample (≤ 200 µL) onto the C18 membrane. Centrifuge at 1,000–1,500 × g for 1–10 minutes, depending on sample volume, passing all solution through.
Optional: For potentially improved retention, reload the flow-through onto the same StageTip and centrifuge again.
Keep the flow-through — do not discard it yet. If something goes wrong, you can check the flow-through for peptide loss to diagnose the problem.
Step 4: Washing
Place the StageTip into a new collection tube. Add 50–100 µL of 0.5% TFA. Centrifuge at 1,000–1,500 × g passing all solution through. Then add 50–100 µL of 0.5% AcOH. Centrifuge at 1,000–1,500 × g passing all solution through.
Optional: Repeat the 0.5% AcOH wash 1–2 more times if your sample contains heavy contaminants. Discard all flow-through.
The acidic wash removes residual salts. The AcOH wash removes salts and TFA in preparation for final elution. For very dirty samples (e.g., high-salt buffers, detergent-containing lysates), additional washes are recommended.
Step 5: Elution
Place the StageTip into a new collection tube (this is your final sample tube — use a low-binding tube). Add 10–30 µL of 80% ACN / 0.5% AcOH onto the C18 membrane. Centrifuge at 1,000–1,500 × g passing all solution through.
Optional: Repeat the elution 1–2 more times for maximum recovery.
At maximum loading capacity (40 µg peptides), up to 100 µL total elution volume across 2–3 elutions may be necessary for quantitative recovery.
After elution: Your desalted peptides are now ready for LC–MS/MS analysis. If needed, concentrate the eluate in a vacuum concentrator (SpeedVac) — but avoid drying completely, as this promotes peptide adsorption to tube surfaces and can reduce recovery. Resuspend or dilute into your LC loading buffer (e.g., 0.1% formic acid, 2–5% ACN).
Troubleshooting
Even experienced proteomics researchers occasionally run into issues with peptide desalting. Here are the most common problems and how to fix them.
Tips for Best Results
1. Never let the membrane dry
This is the single most important rule. During conditioning and equilibration, always leave a thin layer of liquid above the membrane. A dry C18 membrane develops air channels that allow sample to pass through without proper contact, dramatically reducing binding efficiency.
2. Always use a new collection tube for elution
Reusing the same tube from the wash step introduces salt carry-over into your clean eluate. This salt can affect data quality during mass spectrometry acquisition.
3. Save your flow-through
Until you have confirmed that your desalting worked (by running the sample on the mass spec or checking peptide concentration), keep the loading flow-through in case you need to troubleshoot.
4. Match your tip format to your sample amount
The 200 µL, 2-layer C18-HD StageTip can bind up to 40 µg of BSA peptides. For low-input samples (single-cell, LCM, limited biopsies), the 10 µL format (Part No. 70-2019-1019-0) is a better choice. This size minimizes dead volume and surface losses. Make sure to scale buffer volumes proportionally when switching formats.
5. Don't over-dry in the SpeedVac
Completely drying peptides promotes strong adsorption to tube walls, which can reduce recovery. Concentrate to near dryness, then resuspend immediately in your LC loading buffer.
6. Consider your downstream analysis
For LC–MS/MS, resuspend or dilute in 0.1% formic acid with 2–5% ACN. For MALDI-MS, you can spot the eluate directly mixed with matrix solution. Adjust final volume based on your injection amount and desired peptide concentration (typically 0.2–1 µg/µL for LC–MS).
Quick Reference: Workflow Summary
What Else Can You Do with Empore StageTips?
C18-HD desalting is just the starting point. The Empore StageTip portfolio includes seven products covering desalting, fractionation, and integrated workflows:
C8 — Better recovery for hydrophobic peptides and small proteins
SDB-XC — Higher capacity (50 µg) and broader pH tolerance
SDB-RPS — RP and cation mixed mode; No conditioning needed; desalting with optional fractionation
SCX / SAX — Ion-exchange fractionation for deeper proteome coverage
C18–SCX / C18–SCX–C18 — All-in-one desalting + fractionation devices
Not sure which tip is right for your experiment? Contact us and our team will reach out to you shortly.
Conclusion
Peptide desalting with C18 StageTips is a simple, reliable, and cost-effective method that can significantly improve the quality of your LC–MS/MS data. The key to consistent results is careful attention to a few fundamental rules: proper conditioning, acidic sample loading, no drying of the membrane, and clean elution into a fresh tube. With this protocol and the troubleshooting guide above, even researchers new to proteomics can achieve high peptide recovery and clean mass spectrometry data.
1. Rappsilber, J.; Mann, M.; Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2007, 2(8), 1896–1906.
2. Kulak, N.; Pichler, G.; Paron, I. et al. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells. Nat. Methods 2014, 11, 319–324.