Precursor ion scanning for detection and structural characterization of heterogeneous glycopeptide mixtures

Combining source collision-induced dissociation (CID) and tandem mass spectral acquisition in a pseudo-MS 3 experiment using a linear ion trap results in a highly selective and sensitive approach to identifying glycopeptide elution from a protein digest. The increased sensitivity is partially attributed to the nonselective nature of source CID, which allows simultaneous activation of all charge states and coeluting glycoforms generating greater ion abundance for the mass-to-charge (m/z) 204 and/or 366 oxonium ions. Unlike source CID alone, a pseudo-MS 3 approach adds selectivity while improving sensitivity by eliminating chemical noise during the tandem mass spectral acquisition of the oxonium ions in the linear ion trap. Performing the experiments in the hybrid linear ion trap/Fourier transform-ion cyclotron resonance (FT-ICR) enables subsequent high-resolution/high-mass accuracy full-scan mass spectra (MS) and parallel acquisition of MS/MS in the linear ion trap to be completed in 2 s directly following the pseudo-MS 3 scan to collate identification and characterization of glycopeptides in one experimental scan cycle. Analysis of bovine fetuin digest using the combined pseudo-MS 3 , high-resolution MS, and data-dependent MS/MS events resulted in identification of four N-linked and two O-linked glycopeptides without enzymatic cleavage of the sugar moiety or release of the sialic acids before analysis. In addition, over 95% of the total protein sequence was identified in one analytical run. (J Am Soc Mass Spectrom 2006, 17, 168 -179)

Section: Fetuin Analysis Resultsmentioning

confidence: 99%

A novel approach for identification and characterization of glycoproteins using a hybrid linear ion trap/FT-ICR mass spectrometer

Peterman

Mulholland

2006

“…In addition, the signal-to-noise for the scanning experiments was quite low, and several unidentifiable peaks appeared, particularly in the neutral loss scans. While others have proposed these methods as the best way to identify glycopeptides [22], for the samples studied herein these methods were not as effective at identifying all the glycoforms present as the standard MS data. (The scanning experiments, however, are useful because they can provide structural information about the compounds.…”

Section: Glycopeptides Identifiedmentioning

confidence: 89%

“…When glycopeptides undergo CID, an abundant product ion is generated by a cleavage between the first and second GlcNAc adjacent to the peptide [22] as shown in Figure 3. Such a cleavage produces ions containing the GlcNAc attached to whatever peptide was produced by proteolysis.…”

Section: Detection Of Constituent Monosaccharides and Proteolytic Pepmentioning

confidence: 99%

“…These ions were m/z 147 for deoxy-hexose, m/z 163 for Hex, m/z 204 for HexNAc, m/z 274 for the dehydrated sialic acids, and m/z 292 for sialic acid. A glycopeptide, GlcNAc-NIT (GlcNAc is N-acetylglucosamine), at m/z 550 was also selected as a characteristic N-linked glycopeptide product ion, based on previous findings which demonstrate that the cleavage producing this ion occurs readily during collisional activation [22]. No glycopeptides were detected by precursor ion scan of m/z 147, 274, and 292.…”

Section: Parent Ion Scanmentioning

confidence: 99%

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Glycoprotein profiling by electrospray mass spectrometry

Jiang

Desaire

Butnev

et al. 2004

This work compares several different methods of site-specific analysis of glycoproteins using electrospray mass spectrometry. The glycoprotein, oLH␣ (ovine luteinizing hormone, ␣-subunit) was chosen as an appropriate example protein for these studies because of its biological relevance and extreme microheterogeneity. More than 20 unique glycoforms were detected for this glycoprotein at the Asn 56 site of oLH␣. The carbohydrates present at this site affect receptor binding affinity, so understanding the great variety in the composition of these carbohydrates is important in studying ligand binding interactions. MS data was acquired on a quadrupole ion trap, a triple quadrupole, and a quadrupole time of flight mass spectrometer, and carbohydrate composition at the Asn 56 site of oLH␣ was determined using these instruments. Additionally, neutral loss and precursor ion scanning modes were also used to identify the glycoforms present, and these techniques were compared to the standard MS data. Of the three instruments compared in the study, the qTOF mass spectrometer achieved the lowest sample consumption, but all three instruments were useful in profiling the glycopeptide composition. (J Am Soc Mass Spectrom 2004, 15, 750 -758)

“…Information with respect to the compositions and the sequences of the glycans can also be obtained. The characteristic fragment ions used for this purpose are normally the "reporter" oxonium ions of hexose at m/z 163.060, of N-acetylhexosamines at m/z 204.084, and of hexoylhexosamine at m/z 366.139 [3], but much larger oxonium ions have also recently been used [4]. These fragment ions enable the selective detection of glycosylated peptides using precursor ion scanning or skimmer fragmentation routines on triple or single quadrupole mass spectrometers, respectively.…”

mentioning

confidence: 99%

Using optimized collision energies and high resolution, high accuracy fragment ion selection to improve glycopeptide detection by precursor ion scanning

Jebanathirajah

Steen

Roepstorff

2003

Glycosylation is the most widespread protein modification and is known to modulate signal transduction and several biologically important interactions. In order to understand and evaluate the biological role of glycosylation it is important to identify the glycosylated protein and localize the site glycosylation under particular biological conditions. To identify glycosylated peptides from simple mixtures, i.e., in-gel digests from single SDS PAGE bands we performed high resolution, high accuracy precursor ion scanning using a quadrupole TOF instrument equipped with the Q 2 pulsing function. The high resolving power of the quadrupole TOF instrument results in the selective detection of glycan specific fragment ions minimizing the interference of peptide derived fragment ions with the same nominal mass. Precursor ion scanning has been previously described for these glycan derived ions. However the use of this method has been limited by the low specificity of the method. The analysis using precursor ion scanning can be applied to any peptide mixture from a protein digest without having previous knowledge of the glycosylation of the protein. C urrent genome sequencing efforts have resulted in a remarkable resource of protein and putative protein sequences. These protein sequences, which are stored in accessible databases, increase the facility with which proteins may be identified using mass spectrometry. As protein identification at the subpicomole level becomes routine, there is a shift in interest from simple protein identification, i.e., correlation of a sample spot on a PAGE gel with an accession number, to protein characterization with special emphasis on the analysis of the co-and posttranslational protein modifications. One of these modifications is protein glycosylation, which is the most common protein modification. It is estimated that 50% of all proteins are glycosylated [1]. This modification plays a major structural role but is also heavily involved in cell-cell recognition and modulating molecular interaction. Furthermore, there is the increasing notion that reversible glycosylation plays a pivotal role in signaling mechanisms [2]. In order to understand and evaluate the biological role of glycosylation in detail it is important to analyze the glycosylation. A comprehensive analysis comprises three steps, each of them is a challenge on its own: (1) The identification of glycosylated proteins and peptides, (2) the localization of the glycosylation sites, and (3) the elucidation of the glycan structure. One of the major problems with glycosylation analysis is the fact that this modification is normally highly heterogeneous such that it induces a fairly undefined mass shift that can easily exceed the mass of the peptide. In contrast, "simple" modifications such as phosphorylation or acetylation induce well-defined mass shift (or multiples thereof).One approach that can be used to identify glycosylated peptides is precursor ion scanning for glycan derived fragment ions. As further MS/MS studies of the id...