Glycopeptide Enrichment for MALDI-TOF Mass Spectrometry Analysis by Hydrophilic Interaction Liquid Chromatography Solid Phase Extraction (HILIC SPE)

Pia Hønnerup Jensen, Simon Mysling, Peter Højrup, Ole Nørregaard Jensen

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Glycoproteins, and in particular glycopeptides, are highly hydrophilic and are often not retained by reversed phase (RP) chromatography. The separation principle of normal phase (NP) is based on hydrophilic interactions, which in many aspects is complementary to RP separations. Hydrophilic interaction liquid chromatography (HILIC) is a fairly new variation of the NP separations used in the 1970s, the major difference being the use of aqueous solvents. HILIC provides a versatile tool for enrichment of glycopeptides before mass spectrometric (MS) analysis, particularly when used for solid phase extraction (SPE), or in combination with other chromatographic resins or ion-pairing reagents. HILIC SPE can be used for glyco-Profiling, i.e., for determining the glycan heterogeneity at one specific glycosylation site, for enrichment of glycopeptides from a complex mixture of peptides, as well as for pre-fractionation of complex samples at the protein or peptide level. In this chapter we present a straightforward HILIC SPE enrichment technique and then combine C18 RP and HILIC enrichment for analysis of glycopeptides. Finally, we demonstrate HILIC enrichment using trifiuoroacetic acid as an ion-pairing reagent for the enrichment of glycopeptides prior to mass spectrometry analysis.

Original languageEnglish
JournalMethods in Molecular Biology
Volume951
Pages (from-to)131-144
ISSN1064-3745
DOIs
Publication statusPublished - 2013

Fingerprint

Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Solid Phase Extraction
Liquid Chromatography
Ions
Peptides
Reverse-Phase Chromatography
Complex Mixtures
Glycosylation
Acids
Proteins

Keywords

  • Enrichment
  • Glycopeptides
  • HILIC
  • Ion-pairing reagent
  • MALDI-TOF MS
  • N -glycosylation
  • SPE

Cite this

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title = "Glycopeptide Enrichment for MALDI-TOF Mass Spectrometry Analysis by Hydrophilic Interaction Liquid Chromatography Solid Phase Extraction (HILIC SPE)",
abstract = "Glycoproteins, and in particular glycopeptides, are highly hydrophilic and are often not retained by reversed phase (RP) chromatography. The separation principle of normal phase (NP) is based on hydrophilic interactions, which in many aspects is complementary to RP separations. Hydrophilic interaction liquid chromatography (HILIC) is a fairly new variation of the NP separations used in the 1970s, the major difference being the use of aqueous solvents. HILIC provides a versatile tool for enrichment of glycopeptides before mass spectrometric (MS) analysis, particularly when used for solid phase extraction (SPE), or in combination with other chromatographic resins or ion-pairing reagents. HILIC SPE can be used for glyco-Profiling, i.e., for determining the glycan heterogeneity at one specific glycosylation site, for enrichment of glycopeptides from a complex mixture of peptides, as well as for pre-fractionation of complex samples at the protein or peptide level. In this chapter we present a straightforward HILIC SPE enrichment technique and then combine C18 RP and HILIC enrichment for analysis of glycopeptides. Finally, we demonstrate HILIC enrichment using trifiuoroacetic acid as an ion-pairing reagent for the enrichment of glycopeptides prior to mass spectrometry analysis.",
keywords = "Enrichment, Glycopeptides, HILIC, Ion-pairing reagent, MALDI-TOF MS, N -glycosylation, SPE",
author = "Jensen, {Pia H{\o}nnerup} and Simon Mysling and Peter H{\o}jrup and Jensen, {Ole N{\o}rregaard}",
year = "2013",
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language = "English",
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Glycopeptide Enrichment for MALDI-TOF Mass Spectrometry Analysis by Hydrophilic Interaction Liquid Chromatography Solid Phase Extraction (HILIC SPE). / Jensen, Pia Hønnerup; Mysling, Simon; Højrup, Peter; Jensen, Ole Nørregaard.

In: Methods in Molecular Biology, Vol. 951, 2013, p. 131-144.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Glycopeptide Enrichment for MALDI-TOF Mass Spectrometry Analysis by Hydrophilic Interaction Liquid Chromatography Solid Phase Extraction (HILIC SPE)

AU - Jensen, Pia Hønnerup

AU - Mysling, Simon

AU - Højrup, Peter

AU - Jensen, Ole Nørregaard

PY - 2013

Y1 - 2013

N2 - Glycoproteins, and in particular glycopeptides, are highly hydrophilic and are often not retained by reversed phase (RP) chromatography. The separation principle of normal phase (NP) is based on hydrophilic interactions, which in many aspects is complementary to RP separations. Hydrophilic interaction liquid chromatography (HILIC) is a fairly new variation of the NP separations used in the 1970s, the major difference being the use of aqueous solvents. HILIC provides a versatile tool for enrichment of glycopeptides before mass spectrometric (MS) analysis, particularly when used for solid phase extraction (SPE), or in combination with other chromatographic resins or ion-pairing reagents. HILIC SPE can be used for glyco-Profiling, i.e., for determining the glycan heterogeneity at one specific glycosylation site, for enrichment of glycopeptides from a complex mixture of peptides, as well as for pre-fractionation of complex samples at the protein or peptide level. In this chapter we present a straightforward HILIC SPE enrichment technique and then combine C18 RP and HILIC enrichment for analysis of glycopeptides. Finally, we demonstrate HILIC enrichment using trifiuoroacetic acid as an ion-pairing reagent for the enrichment of glycopeptides prior to mass spectrometry analysis.

AB - Glycoproteins, and in particular glycopeptides, are highly hydrophilic and are often not retained by reversed phase (RP) chromatography. The separation principle of normal phase (NP) is based on hydrophilic interactions, which in many aspects is complementary to RP separations. Hydrophilic interaction liquid chromatography (HILIC) is a fairly new variation of the NP separations used in the 1970s, the major difference being the use of aqueous solvents. HILIC provides a versatile tool for enrichment of glycopeptides before mass spectrometric (MS) analysis, particularly when used for solid phase extraction (SPE), or in combination with other chromatographic resins or ion-pairing reagents. HILIC SPE can be used for glyco-Profiling, i.e., for determining the glycan heterogeneity at one specific glycosylation site, for enrichment of glycopeptides from a complex mixture of peptides, as well as for pre-fractionation of complex samples at the protein or peptide level. In this chapter we present a straightforward HILIC SPE enrichment technique and then combine C18 RP and HILIC enrichment for analysis of glycopeptides. Finally, we demonstrate HILIC enrichment using trifiuoroacetic acid as an ion-pairing reagent for the enrichment of glycopeptides prior to mass spectrometry analysis.

KW - Enrichment

KW - Glycopeptides

KW - HILIC

KW - Ion-pairing reagent

KW - MALDI-TOF MS

KW - N -glycosylation

KW - SPE

U2 - 10.1007/978-1-62703-146-2_10

DO - 10.1007/978-1-62703-146-2_10

M3 - Journal article

VL - 951

SP - 131

EP - 144

JO - Methods in Molecular Biology

JF - Methods in Molecular Biology

SN - 1064-3745

ER -