Agilent Community
Agilent Community
  • User
  • Site
  • Search the Community
  • User
Consumables
  • Technical Areas
Consumables
Wiki Sample Prep Pointers - Peptide Mapping Part I
  • Announcements
  • Forum
  • Files
  • Wiki
  • More
  • Cancel
  • Consumables
  • A Beginner’s Guide to Hydrophobic Interaction Chromatography
  • A simple tip to protect your columns
  • A Tip for Preparing Robust and Consistent Mobile Phases
  • Achieve accurate and repeatable gas flow meter measurements
  • AdvanceBio Columns Blog Series
  • Agilent 123 Meter Sodium probe maintenance
  • Agilent Collection of Columns, Supplies, and Standards Resources
  • Analysis of microplastics in the environment
  • Analyzing ADCs by HIC
  • Avoiding downtime in the lab: top tips for GC/MS success
  • Best Practices for Aqueous Mobile Phases
  • Best Practices for Making Good Connections
  • Bio LC Column User Guides
  • Bursting Tubing and Columns (GC and HPLC)
  • Calculate the Maximum Allowable Working Pressure for Tubing
  • Calculating Column Volume
  • Cannabis Potency Testing: a Reliable, Cost-Effective Method
  • Carbohydrate Analyses in LC
  • Checking Your Intuition - Sub 2 µm vs Superficially Porous
  • Choosing the right GC Injection Technique
  • Choosing the right pore size for size exclusion chromatography
  • Columns, Supplies, and Standards Knowledgebase
  • Consumables Applications and Workflows
  • Consumables Recommended Supplies Lists for Agilent.com
  • Custom Product Request
  • Extreme Makeover – Derivatizations in Chromatography – Part 1 GC
  • Extreme Makeover – Derivatizations in Chromatography – Part 2 LC
  • Fake It Until You Make It: When BioInert Isn’t an Option
  • Flipping Amino Acid Analysis on Its Head
  • Glycans at a glance:  Analyzing therapeutic glycoproteins
  • Handle and Care of Syringes
  • Help! My Peaks Look Strange - Fronting and Tailing in GC
  • Help! My Peaks Look Strange - Saddle Points - LC/GC Troubleshooting
  • How do I select a Split/Splitless liner?
  • Hydrophobic Interaction Chromatography of Proteins and mAbs
  • Importance of Silica Particle Strength for Sub-2 µm SEC Columns
  • KB: Ferrules recommended for GC self-tightening column nut
  • LC and LC/MS Columns - USP Designations
  • LC Column User Guides
  • LC Method Translation - the Dwell Volume
  • Minimize spectroscopy workflow disruptions
  • Minimizing Metals for Best HILIC Results
  • More than just a drink: Analyzing the elemental composition of beer
  • Multi-Attribute Methods – Peptide Mapping Part IV
  • Must See Webinars
  • Nomenclature of CFC's/Freons/Halons/Coolants
  • Oligonucleotide Analysis - Unexpected Details Matter
  • Optimizing Bonding Chemistry for Sub-2 µm SEC Particles
  • Pass the Salt, Please – Mobile Phase Preparation for HIC
  • Pesticides and their stability during GC analyses
  • Pre-Columns - the forgotten art of using retention gaps
  • Problematic polar analytes? Hello HILIC…
  • Protecting your laboratory productivity
  • Recommended Reading
  • Sample Prep Pointers - Peptide Mapping Part I
  • Save your results with sample filtration
  • Simplified cone inspection with the new Agilent LED measuring magnifier
  • Software - Supported Method Development - The Scanview Application
  • Software tool for the ADM Flow Meter (G6691A)
  • Stay Safe: A Win-Win for Solvent Storage
  • Streamline your sample processing
  • The importance of chemical composition for vial performance
  • Tips & Tricks for Amino Acid Analysis – Part I
  • Tips & Tricks for Amino Acid Analysis – Part II
  • Tips & Tricks for Amino Acid Analysis – Part III
  • Tips & Tricks for Amino Acid Analysis – Part IV
  • Tips for Smooth Sailing with HIC
  • Troubleshooting HPLC autosamplers
  • Troubleshooting HPLC degassers
  • Troubleshooting Sequence Coverage – Peptide Mapping Part III
  • UltiMetal Plus Flexible Metal Ferrule
  • UV, MS, TFA, and Formic Acid – What to use? Peptide Mapping Part II
  • What are the typical % Gain or EHT values for hollow cathode lamps?
  • You Need Lamps or Chemical Standards for Atomic Absorption Single-Element Analyses?
Still Need Help?

Post your question in our User Forum or Contact Support.

Sample Prep Pointers - Peptide Mapping Part I

Created by anne_blackwell anne_blackwell over 2 years ago | Last modified by Agilent Agilent over 2 years ago

Careful and consistent sample preparation is the first step to quality peptide mapping data.

 

Protein digestion for peptide mapping is among the most complicated sample prep to be done in when monitoring critical quality attributes – perhaps rivaled only by sample prep for glycan analysis.  Agilent’s Peptide Mapping “How to” Guide lays out a general protocol, but there are always proteins or matrices that need special consideration.  There are multiple steps, each with choices to be made and opportunity for optimization.  Putting some thought into your sample prep procedure can make a big difference in the separation and detection of your peptides.  I’ve quizzed a few colleagues (thanks, Suresh, Veronica, and Rita!), and based on our collective experience we’ve come up with these tips. 

  

Sample Preparation

Some matrices require clean up or adjustments for more efficient digestion.  An affinity capture step can be used to either deplete matrix proteins such as albumin and immunoglobulins or to enrich the target protein based on PTMs such as phosphorylation or glycosylation.

  • Think about your whole workflow early on – if you’re planning to use MS for detection, think carefully about the reagents you use and how much you use. How easy are they to remove from the sample in a clean up step?  Will they lead to ion suppression?
  • Consider taking a blank sample through your entire process as a control for troubleshooting purposes (sample prep through any enrichment and clean up).
  • pH can be a balancing act – lower pH can help prevent deamidation, but many proteases work more efficiently at slightly elevated pH. (Don’t you hate choosing the lesser of two evils?)

 

Denaturation, Reduction, and Alkylation

For the protease to efficiently access cleavage sites, its necessary to denature the protein and reduce and alkylate the disulfide bonds (unless of course you’re looking to map the disulfide bonds).

  • If you’re using urea to denature, don’t heat the sample! This will lead to lysine carbamylation, and we don’t (usually) want to add any PTMs.
  • Guanidine can inhibit protease activity, so be sure to dilute or remove it prior to digestion.
  • Highly concentrated or complex proteins can be prone to precipitation during the denaturation step, so keep an eye out. Dilution before denaturation may help.
  • DTT and mercaptoethanol are really stinky reductants. TCEP is just as effective as DTT, and it’s odorless (a good enough reason in my book!).  Just beware it’s not particularly stable in phosphate buffer at neutral pH, so a fresh solution is necessary.

 

Digestion

Trypsin is the most commonly used protease since it’s cleavages are so well defined and predictable, but there are a variety of digestion agents available if trypsin’s lysine and arginine cleavages don’t produce suitable peptides for separation and detection.  We’ll talk more about this in a future post.  The optimum pH is dependent on the chosen digestion reagent, but digestion time, temperature, and protease concentration are variables that are independent on one another as well as the target protein and protease.

  • Trypsin cleavage is slower when Lys or Arg are adjacent to Cys, Glu, or Asp, and doesn’t occur at all when Lyr or Arg is followed by Pro.1
  • Two separate additions of trypsin may help achieve complete digestion.
  • After digestion, acidify the sample with 0.1% formic acid rather than 1%.
  • Many protocols proscribe a certain incubation or reaction time, but if you’re implementing a protocol to use long-term, it would be worth an experiment to see how long that step really takes. Many a grad student (yours truly included) has let a reaction go for 12 hours because they wanted to go home for the night!
  • Trypsin (or other protease) autolysis can lead to extra peaks in your peptide map. Some trypsin products are more prone to this than others.  Just something to keep in the back of your mind if you’re troubleshooting coelution or chasing unidentified peaks (something your blank control can help with).

Trypsin is so prevalent that protocols are plentiful, but while we’re on the topic, I wanted to mention this Nature Protocols article by Giansanti et al that details digestion protocols for six other proteases along with discussion of the pros and cons of each.1

 

Enrichment and Clean Up

If you’re looking for specific PTMs such as phosphorylation or glycosylation, enrichment following digestion may be helpful.  Clean up is often necessary for successful analysis of your peptide map, especially with MS detection as salts can significantly limit ionization and therefore sensitivity.

  • Desalt the sample before MS analysis for best sensitivity. Salt can significantly limit how well the sample ionizes.
  • If the sample appears opaque or cloudy, filter it before injection.

 

A final general tip – once you have settled on a protocol, stick to it!  Any variation from the protocol can lead to inconsistent results and more questions.

 

Talk soon –

Anne

 

References

  1. Giansanti, L. Tsiatsiani, T.Y. Low, A.J.R. Heck. Six alternative proteases for mass spectrometry-based proteomics beyond trypsin.  Nature Protocols, 2016.

 

Keywords: Bio columns, liquid chromatography, tips and tricks, peptide mapping, sample prep, AdvanceBio blog

  • tips and tricks
  • peptide mapping
  • bio columns
  • advancebio blog
  • Biopharma LC Columns & Consumables
  • sample prep
  • liquid chromatography
  • Share
  • History
  • More
  • Cancel
Anonymous
Related

Agilent Community Feedback

Agilent Community Feedback

×
We are glad this was helpful! We are sorry this was not helpful. If you still need assistance please create a community post or contact support. To help us improve, please provide any additional feedback. For full details of how we will treat your information, please view our privacy policy.
Submit Cancel
Submit Cancel
Recommended
Privacy Statement
Terms of Use
Contact Us
Site Help