Peptide Separation

Divide and conquer

Strong cation-exchange (SCX) chromatography

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SCX has been used extensively for the fractionation of proteins and peptides based on charge. The SCX stationary phase usually contains aliphatic sulfonic acid groups that are negatively charged in aqueous solution, therefore tightly binding any strongly basic analytes. To recover the analyte, the resin is then washed with a solvent neutralizing this ionic interaction. Most tryptic peptides in acidic pH are characterized by a net charge of +2 and above, and they can be therefore separated by SCX from peptides possessing a net charge of +1, such as trypsin-generated phosphopeptides, C-terminal peptides, or peptides with blocked N-termini (i.e., peptides with blocked N-terminal free amine group, for instance, by N-acetylation), as well as from peptides containing higher charges, including ones containing missed cleavages and therefore more arginine and lysine residues. SCX fractionation can also be performed in a solid-phase extraction cartridge format for a rapid but lower resolution fractionation. SCX can also be conducted in microscale solid-phase extraction format (i.e. after IMAC) by utilizing a stage tip packed with an SCX disk.
  • Fractionation of peptides by strong cation-exchange liquid chromatography. Chan KC, Issaq HJ. Methods Mol Biol. 2013;1002:311-5. link
  • Strong cation exchange chromatography in analysis of posttranslational modifications: innovations and perspectives. Edelmann MJ. J Biomed Biotechnol. 2011;2011:936508. link
  • A solid phase extraction-based platform for rapid phosphoproteomic analysis. Dephoure N, Gygi SP. Methods. 2011 Aug;54(4):379-86 link
  • Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Rappsilber J, Mann M, Ishihama Y. Nat Protoc. 2007;2(8):1896-906 link

Avidin

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Isolation of biotin labeled peptides
  • Check out this biotinylation overview at Pierce link
  • Pierce™ Cell Surface Biotinylation and Isolation Kit link


Spatial in-vivo biotinylation strategies
  • Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2. Hung V, Udeshi ND, Lam SS, Loh KH, Cox KJ, Pedram K, Carr SA, Ting AY Nat Protoc. 2016 Mar;11(3):456-75. link
  • Biotinylation by antibody recognition-a method for proximity labeling. Bar DZ, Atkatsh K, Tavarez U, Erdos MR, Gruenbaum Y, Collins FS. Nat Methods. 2018 Feb;15(2):127-133. link
  • Direct detection of biotinylated proteins by mass spectrometry. Schiapparelli LM, McClatchy DB, Liu HH, Sharma P, Yates JR 3rd, Cline HT. J Proteome Res. 2014 Sep 5;13(9):3966-78. link

Phosphopeptide enrichment with IMAC (Immobilized Metal Affinity Chromatography)

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To achieve robust MS results, enrichment of phosphopeptide samples is essential because of low abundance and poor ionization relative to non-phosphorylated peptides. Phosphopeptide enrichment reduces sample complexity and enables effective identification and characterization of phosphorylated peptides by MS.

A practical recipe to survey phosphoproteomes.
Edelman WC, Haas KM, Hsu JI, Lawrence RT, Villén J. Methods Mol Biol. 2014;1156:389-405. doi: 10.1007/978-1-4939-0685-7_26. link

  • Thermo Pierce Fe-NTA Phosphopeptide Enrichment Kit enables fast and efficient enrichment of phosphorylated peptides to process protein digests or strong cation-exchange peptide fractions for analysis by mass spectrometry

  • Thermo Pierce TiO2 Phosphopeptide Enrichment and Clean-up Kit enables fast, selective enrichment of phosphorylated peptides for mass spectrometry, it complements the Pierce Fe-NTA IMAC Phosphopeptide Enrichment Kit

  • Sigma PHOS-Select Iron Affinity Gel is an Iron [Fe(III)] chelate matrix based on Sigma's NTA analog chelate ligand. The matrix provides high capacity affinity binding of molecules containing phosphate groups

  • Sigma PHOS-Select Gallium Silica Spin Column Kit is a capture matrix is a Ga3+ chelate silica based on a proprietary nitriloacetic acid (NTA) analog

  • The Pierce Graphite Spin Columns improve phosphopeptide analysis by efficiently binding hydrophilic peptides and efficiently removing urea, salts and other contaminants before MS analysis. The C18 resins and C18 tips that are commonly used to desalt peptides are excellent for use with hydrophobic peptides but do not efficiently capture hydrophilic peptides, like phosphopeptides, resulting in enrichment of only hydrophobic fragments.

Glycopeptide

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  • This review covers several glycoprotein/peptide enrichment strategies:
    Quantitative mass spectrometric analysis of glycoproteins combined with enrichment methods. Ahn YH, Kim JY, Yoo JS. Mass Spectrom Rev. 2015 Mar-Apr;34(2):148-65. doi: 10.1002/mas.21428. Epub 2014 Jun 2. link
  • Solid Phase Extraction of N-linked Glycopeptides Using Hydrazide Tip. Jing Chen, Punit Shah, and Hui Zhang Anal Chem. 2013 Nov 19;85(22):10670-4. doi: 10.1021/ac401812b. Epub 2013 Oct 30. link

  • Thermo aminoxyTMTsixplex Label Reagents
    The carbonyl-reactive Thermo Scientific™ aminoxyTMT™ (Tandem Mass Tag™) Label Reagents enable multiplexed characterization and quantitation of carbonyl-containing biomolecules (carbohydrates, steroids, oxidized proteins) by mass spectrometry (MS).

Peptide cleanup protocols for LC-MS

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  • C18 Columns & Peptide Desalting for Mass Spectrometry
    After isolation of peptides, salts and buffers can be removed using reversed phase (RP) resins, of which the C18 matrix is the most ideal for the capture of hydrophobic peptides. The peptides bind to reverse-phase columns in high-aqueous mobile phase, salts and buffers are washed off, and the peptides are eluted using a high-organic mobile phase.

  • ZipTip Pipette Tips
    ZipTip is a 10 µL pipette tip with a 0.6 or 0.2 µL bed of chromatography media fixed at its end with no dead volume. It is ideal for concentrating and purifying samples for sensitive downstream analyses.

  • Omix Tips
    Bond Elut OMIX pipette tips reliably purify and enrich femtomole and picomole levels of peptides and proteins prior to MALDI-TOF or LC/MS/MS. This unique monolithic sorbent consistently delivers uniform flow and strong analyte-to-surface interactions.

  • Protemics sample prep: S-Trap
    S-Trap™ sample processing begins with sample lysis and solubilization in 5% SDS. Proteins are further denatured by acidification to pH < 1 and subsequent exposure to a high concentration of methanol.
    Reduction and alkylation and digestion are performed within the physical confinement of the submicron pores of the trap forcing substrate and protease interaction to yield rapid digestion
    The trap does not have affinity for peptides, which elute after digestion.

  • Protemics sample prep: ProTrap XG
    The ProTrap XG is a dual-stage, disposable filtration and extraction cartridge can help:
    Remove a high level of SDS
    Perform filtration, precipitation, and digestion in a single device

  • Nestgroup Desalting C18 RP MicroSpin column protocol (pdf)
    These spin columns (pdf) will retain non-polar solutes such as peptides (C18) , proteins (C4), and detergents. Salts, and polar solutes like DNA will not be retained
    SUM SS18V 2-100 μl or 3-30 μg
    SEM SS18V 5-200 μl or 5-60 μg
    SMM SS18V 50-450 μl or 30-300 μg

  • Nestgroup Desalting C18 RP MicroSpin (Targa) column protocol (pdf)
    These spin columns (pdf) of water wettable TARGA C18 will retain polar & non-polar solutes such as carbohydrates, nucleotides, polar peptides as well as metabolites and pharmaceutical compounds. Salts will not be retained. This permits the removal of salt from samples prior to mass spectrometry
    SUM SS18R 2-25 μl or 3-30 μg
    SEM SS18R 5-50 μl or 6-60 μg
    SMM SS18R 50-150 μl or 30-300 μg

  • Waters Sep-Pak (WAT054955) for peptide desalting
    Sep-Pak C18 Vac cartridges contain a hydrophobic, reverse-phase, silica-based bonded phase that is used to adsorb analytes of even weak hydrophobicity from aqueous solutions.

  • Detergent Removal from Peptides
    The Thermo Scientific Pierce Detergent Removal Resins are provided in convenient spin-column or plate formats that quickly and efficiently remove ionic, nonionic, and/or zwitterionic detergents from protein or peptide samples to improve compatibility with downstream applications.

  • ThermoTechTip19 for detergent removal (pdf)
  • ThermoPierce Detergent Removal for low-concentration samples (pdf)
  • ThermoPierce Detergent Removal for samples with proteins or peptides at greater than 100μg∕mL (pdf)

  • Nestgroup Hydrophilic Interaction and Detergent Removal (pdf)
    HILIC spin columns (pdf) will retain polar solutes such as peptides, proteins, and polar metabolites. Salts, detergents, and non polar solutes will not be retained. This permits the removal of nonvolatile components from samples prior to mass spectrometry
    SUM HIL 2-25 μl or 3-30 μg
    SEM HIL 5-50 μl or 6-60 μg
    SMM HIL 50-150 μl or 30-300 μg

  • Nestgroup Strong Cation Exchange: post iTRAQ clean-up (pdf)
    These spin columns (pdf) will retain cationic solutes such as peptides, protein digests, or simple organic amines. Desalt samples prior to mass spectrometry.
    SUM HIL-SCX 2-25 μl or 5-50 μg
    SEM HIL-SCX 5-50 μl or 10-100 μg
    SMM HIL-SCX 50-150 μl or 50-500 μg

    Peptide separation techniques