PEPTIDE MAPPING

Strategies to Achieve High Sequence Coverage

LC-MS is frequently used for the purpose of peptide mapping, since digested cell lysate is often too complex for direct mass spectrometric analysis. Ideally all peptides of a digested protein or protein mixture should be identified by their MS/MS spectrum and retention time. Over time, different analytical approaches have been developed and introduced, but reversed phase (RP) chromatography is most commonly used. In many cases the resolving power of a single RP column is not sufficient to separate and identify all peptides present, wherefore alternative columns or additional selectivities are sought.

Complementary HPLC columns based on the techniques of reversed phase (RPLC) and hydrophilic interaction (HILIC) chromatography, together allow a much higher sequence coverage compared to traditional one dimensional RPLC approaches. Several studies have shown that HILIC combined with RPLC can be considered as the most orthogonal combination of separation techniques providing the highest coverage of a 2D separation space.

The zwitterionic ZIC®-HILIC stationary phases have been used for such purposes, and it has been shown that separation is dependent on buffer pH. Compared to strong cation exchange chromatography (SCX), ZIC®-HILIC gives better chromatographic resolution and absence of clustering of charged peptides. At low pH, ZIC®-HILIC separation allows best orthogonality with RP and resembles conventional SCX separation. However, at neutral or slightly basic pH, it enables better chromatography, resulting in more comprehensive data acquisition.

Since HILIC eluents typically contain a high percentage of organic solvent hydrophobic peptides are, additionally, potentially less prone to precipitation during sample handling on the column than in RP chromatography. This means that HILIC columns, despite being mostly applied for analysis of hydrophilic molecules, can be used to identify hydrophobic peptides not detected with a RP column. Another feature using HILIC is that higher sensitivity can be expected for many peptide sequences, due to the high percentage organic content in the mobile phase which enhances volatilisation in the detector.

Below, the potential of using HILIC for peptide mapping is illustrated with three trypsin digested proteins, namely Bovine Serum Albumin (BSA), Cytochrome C, and Ovalbumin. The overall result one can expect when combining with classical approaches, is a higher sequence coverage, thus better characterisation of the proteins. 

Bovine Serum Albumin (BSA)

• HILIC-MS Total Ion Chromatogram
• Identified Peptide Sequences HILIC_vs_RPLC
• Peptide Sequences and Trypsin cleavage patterns

Cytochrome C

• HILIC-MS Total Ion Chromatogram
• Identified Peptide Sequences HILIC_vs_RPLC
• Peptide Sequences and Trypsin cleavage patterns

Ovalbumin

• HILIC-MS Total Ion Chromatogram
• Identified Peptide Sequences HILIC_vs_RPLC
• Peptide Sequences and Trypsin cleavage patterns

Application Notes

• ZIC®-pHILIC Separation of Angiotensin II, Bradykinin and Gly-His-Lys
• ZIC®-HILIC Separation of Gly-His-Lys

Technical Application Notes

•  ZIC®-HILIC for Multidimensional Protein Identification Technology

Scientific References  

1. A New Strategy for Identification of N-Glycosylated Proteins and Unambiguous Assignment of Their Glycosylation Sites Using HILIC Enrichment and Partial Deglycosylation
   P. Hägglund, J. Bunkenborg, F. Elortza, O. Nørregaard Jensen, P. Roepstorff
   J. Proteome Res., 3 (2004) 556-566
    
2. Separation of Isomeric 2-Aminopyridine Derivatized N-Glycans and N-glycopeptides of Human Serum Immunoglobulin G by Using a Zwitterionic Type of Hydrophilic-Interaction Chromatography
   Y. Takegawa, K. Deguchi, T. Keira, H. Ito, H. Nakagawa, S.-I. Nishimura
   J. Chromatogr. A, 1113 (2006) 177-181
    
3. Metabolic Fingerprinting of Rat Urine by LC/MS: Part 1. Analysis by Hydrophilic Interaction Liquid Chromatography-Electrospray Ionization Mass Spectrometry
   H. Idborg, L. Zamani, P.-O. Edlund, I. Schuppe-Koistinen, S. P. Jacobsson
   J. Chromatogr. B, 828 (2005) 9-13
     
4. Metabolic Fingerprinting of Rat Urine by LC/MS: Part 2. Data Pretreatment Methods for Handling of Complex Data
   H. Idborg, L. Zamani, P.-O. Edlund, I. Schuppe-Koistinen, S. P. Jacobsson
   J. Chromatogr. B, 828 (2005) 14-20
    
5. Isolation and Characterization of Glycosylphosphatidylinositol-Anchored Peptides by Hydrophilic Interaction Chromatography and MALDI Tandem Mass Spectrometry
   M. J. Omaetxebarria, P. Hägglund, F. Elortza, N. M. Hooper, J. M. Arizmendi, Ole Nørregaard Jensen
   Anal. Chem., 78 (2006) 3335-3341
    
6. Simple Separation of Isomeric Sialylated N-Glycopeptides by a Zwitterionic Type of Hydrophilic Interaction Chromatography
   Y. Takegawa, K. Deguchi, H. Ito, T. Keira, H. Nakagawa, S.-I. Nishimura
   J. Sep. Sci., 29 (2006) 2533-2540
    
7. Direct Structural Assignment of Neutral and Sialylated N-Glycans of Glycopeptides using Collision-Induced Dissociation MSn Spectral Matching
   H. Ito, Y. Takegawa, K. Deguchi, S. Nagai, H. Nakagawa, Y. Shinohara , S.-I. Nishimura
   Rapid. Commun. Mass Spectrom., 20 (2006) 3557-3565
    
8. Structural Assignment of Disialylated Biantennary N-Glycan Isomers Derivatized with 2-Aminopyridine using Negative-ion Multistage Tandem Mass Spectral Matching
   H. Ito, K. Yamada, K. Deguchi, H. Nakagawa, S.-I. Nishimura
   Rapid. Commun. Mass Spectrom., 21 (2007) 212-218
    
9. Evaluation and Optimization of ZIC-HILIC-RP as an Alternative MudPIT Strategy
   P. J. Boersema, N. Divecha, A. J. R. Heck, S. Mohammed
   J. Proteome Res., 6 (2007) 937-946
    
10. HPLC Columns Partition by Chemometric Methods Based on Peptides Retention
    B. Buszewski, S. Kowalska, T. Kowalkowski, K. Rozpędowska, M. Michel, T. Jonsson
    J. Chromatogr. B, 845 (2007) 253-260
     
11. An Enzymatic Deglycosylation Scheme Enabling Identification of Core Fucosylated N-Glycans and O-Glycosylation Site Mapping of Human Plasma Proteins
    P. Hägglund, R. Matthiesen, F. Elortza, P. Højrup, P. Roepstorff, O. Nørregaard Jensen, J. Bunkenborg
    J. Proteome Res., 6 (2007) 3021-3031
     
12. Barley Peroxidase Isozymes Expression and Post-Translational Modification in Mature Seeds as Identified by Two-Dimensional Gel Electrophoresis and Mass Spectrometry
    S. Laugesena, K. Sass Bak-Jensen, P. Hägglund, A. Henriksen, C. Finnie, B. Svensson, P. Roepstorff
    Int. J. Mass Spectrom., 268 (2007) 244-253
      
13. Secreted Glycoprotein from Live Zaire ebolavirus-Infected Cultures: Preparation, Structural and Biophysical Characterization, and Thermodynamic Stability
    L. G. Barrientos, A. M. Martin, R. M. Wohlhueter, P. E. Rollin
    J. Infect. Des., 196 (2007) (Suppl. 2) S220-S231
     
14. 2D LC Separation and Determination of Bradykinin in Rat Muscle Tissue Dialysate with On-Line SPE-HILIC-SPE-RP-MS
    S. R. Wilson, M. Jankowski, M. Pepaj, A. Mihailova, F. Boix, G. V. Truyols, E. Lundanes, T. Greibrokk
    Chromatographia, 66 (2007) 469-474
     
15. Comparative Evaluation of High-Performance Liquid Chromatography Stationary Phases Used for the Separation of Peptides in Terms of Quantitative Structure-Retention Relationships
    M. Michel, T. Baczek, S. Studzinska, K. Bodzioch, T. Jonsson, R. Kaliszan, B. Buseeski
    J. Chromatogr. A, 1175 (2007) 49-54
     
16. Hydrophilic Interaction Chromatography for Mass Spectrometric Metabonomic Studies of Urine
    S. Cubbon, T. Bradbury, J. Wilson, J. Thomas-Oates
    Anal. Chem., 79 (2007) 8911-8918
     
17. Investigating the Biomarker Potential of Glycoproteins using Comparative Glycoprofiling - Application to Tissue Inhibitor of Metalloproteinases-1
    M. Thaysen-Andersen, I. B. Thøgersen, U. Lademann, H. Offenberg, A. M. B. Giessing, J. J. Enghild, H. J. Nielsen, N. Brünner, P. Højrup
    Biochim. Biophys. Acta, 1784 (2008) 455-463
     
18. Hydrophilic Interaction Liquid Chromatography (HILIC) in Proteomics [Review]
    P. J. Boersema, S. Mohammed, A. J. R. Heck
    Anal. Bioanal. Chem., (2008) online open access
     
19. Improving the Resolution of Neuropeptides in Rat Brain with On-Line HILIC-RP Compared to On-line SCX-RP
    A. Mihailova, H. Malerød, S. R. Wilson, B. Karaszewski, R. Hauser, E. Lundanes, T. Greibrokk
    J. Sep. Sci., 31 (2008) 459-467 
     
20. Two-Dimensional Hydrophilic Interaction Chromatography Coupling Anion Exchange and Hydrophilic Interaction Columns for Separation of 2-Pyridylamino Derivatives of Neutral and Sialylated N-Glycans.
    K. Deguchi, T. Keira, K. Yamada, H. Ito, Y. Takegawa, H. Nakagawa, S.-I. Nishimura
    J. Chromatogr. A, 1189 (2008) 169-174
     
21. Stationary Phases for Hydrophilic Interaction Chromatography, their Characterization and Implementation into Multidimensional
    Chromatography Concepts
    P. Jandera
    J. Sep Sci., 31 (2008) 1421-1437 
     
22. Chromatographic Deuterium Isotope Effects of Derivatized N-Glycans and N-Glycopeptides in a Zwitterionic Type of Hydrophilic Interaction Chromatography.
    Y. Takegawa, M. Hatou, K. Deguchi, H. Nakagawa, S.-I. Nishimura
    J. Sep. Sci., 31 (2008) 1594-1597
     
23. Profiling of N- and O-Glycopeptides of Erythropoietin by Capillary Zwitterionic-Type of Hydrophilic Interaction Chromatography/Electrospray Ionization Mass Spectrometry.
    Y. Takegawa, H. Ito, T. Keira, K. Deguchi, H. Nakagawa, S.-I. Nishimura
    J. Sep. Sci., 31 (2008) 1585-1593 
      
24. Metabolomic Profiling of Drosophila Using Liquid
    Chromatography Fourier Transform Mass Spectrometry
    M.A. Kamleh, Y. Hobani, J.A.T. Dow, D.G. Watson
    FEBS Letters, 582 (2008) 2916-2922
     
25. Metabolomic Profiling using Orbitrap Fourier Transform Mass Spectrometry with Hydrophilic Interaction Chromatography: A Method with Wide Applicability to Analysis of Biomolecules
    A. Kamleh, M. P. Barrett, D. Wildridge, R. J. S. Burchmore, R. A. Scheltema, D. G. Watson
    Rapid Commun. Mass Spectrom., 22 (2008) 1912-1918
      
26. Assessment of Lectin and HILIC Based Enrichment Protocols for Characterization of Serum Glycoproteins by Mass Spectrometry
    Cosima D. Calvano, Carlo G. Zambonin, Ole N. Jensen
    J. Proteomics, 71 (2008) 304-317
      
27. Quantitative Site-Specific Analysis of Protein Glycosylation by LC-MS Using Different Glycopeptide Enrichment Strategies
    J. Wohlgemuth, M. Karas, T. Eichhorn, R. Hendriks, S. Andrecht
    Anal. Biochem., (2009), in press available online August 21
     
28. Glycopeptide profiling of beta-2-glycoprotein I by mass spectrometry reveals attenuated sialylation in patients with antiphospholipid syndrome
    A. Kondo, T. Miyamoto, O. Yonekawa, A. M. Giessing, E. C. Østerlunda, O. N. Jensen
    J. Proteomics (2009), in press