HILIC and ERLIC Enrichment of Glycopeptides Derived from Breast and Brain Cancer Cells

Zacharias, Lauren G.; Hartmann, Alyssa K.; Song, Ehwang; Zhao, Jingkun; Zhu, Rui; Mirzaei, Parvin; Mechref, Yehia

doi:10.1021/acs.jproteome.6b00429

Cited by 84 publications

(79 citation statements)

References 36 publications

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“…Several methods and bioinformatics approaches based on additional qualitative information have been developed to improve the identification of glycopeptides . The most common is the use of parallel analysis of glycopeptides trimmed or deglycosylated by enzymatic treatment such as PNGase F . Chen et al.…”

Section: Resultsmentioning

confidence: 99%

“…Several methods and bioinformatics approaches based on additional qualitative information have been developed to improve the identification of glycopeptides [26]. The most common is the use of parallel analysis of glycopeptides trimmed or deglycosylated by enzymatic treatment such as PNGase F [27][28][29]. Chen et al reported an approach based on the reduction of the glycan structures to a mannose core that resulted in an increased confidence of the glycopeptide assignments [27].…”

Section: Prediction Of the Retention Time Windowsmentioning

confidence: 99%

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Study of structure‐dependent chromatographic behavior of glycopeptides using reversed phase nanoLC

2017

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Analysis of glycosylation is challenging due to micro- and macro-heterogeneity of the protein attachment. A combination of liquid chromatography with tandem mass spectrometry is one of the most powerful tools for glycopeptide analysis. In this work, we show the effect of various monosaccharide units on the retention time of glycopeptides. Retention behavior of several glycoforms of six peptides obtained from tryptic digest of haptoglobin, hemopexin and sex hormone binding globulin was studied on a reversed phase chromatographic column. We observed reduction of the retention time with increasing number of monosaccharide units of glycans attached to the same peptide backbone. Fucosylation of larger glycans provides less significant retention time shift than for smaller ones. Retention times of glycopeptides were expressed as relative retention times (RRT). These relative retention times were used for calculation of upper and lower limits of glycopeptide retention time windows under the reversed phase conditions. We then demonstrated on the case of a glycopeptide of haptoglobin that the predicted retention time window boosts confidence of identification and minimizes false positive identification. Relative retention time,as a qualitative parameter, is expected to improve LC-MS/MS characterization of glycopeptides.

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Section: Resultsmentioning

confidence: 99%

Section: Prediction Of the Retention Time Windowsmentioning

confidence: 99%

Study of structure‐dependent chromatographic behavior of glycopeptides using reversed phase nanoLC

2017

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“…Dynamic exclusion was enabled to exclude ions repeated within 20 seconds. The energy used for HCD was 45% normalized collision energy optimized in previous studies (75–77) while 30% normalized collision energy was used for CID which was also previously optimized (78). A mass tolerance of 10 ppm was used for extracted ion chromatograms (EICs) of full MS data.…”

Section: Methodsmentioning

confidence: 99%

LC-MS/MS analysis of permethylated N-glycans facilitating isomeric characterization

et al. 2016

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The biosynthesis of glycans is a template free process, hence compositionally identical glycans may contain highly heterogeneous structures. Meanwhile, the functions of glycans in biological processes are significantly influenced by the glycan structure. Structural elucidation of glycans is an essential component of glycobiology. Although NMR is considered the most powerful approach for structural glycan studies, it suffers from low sensitivity and requires highly purified glycans. Although mass spectrometry (MS) based methods have been applied in numerous glycan structure studies, there are challenges in preserving glycan structure during ionization. Permethylation is an efficient derivatization method that improves glycan structural stability. In this report, permethylated glycans are isomerically separated, thus facilitating structural analysis of a mixture of glycans by LC-MS/MS. Separation by porous graphitic carbon liquid chromatography at high temperatures in conjunction with tandem mass spectrometry (PGC-LC-MS/MS) was utilized for unequivocal characterization of glycan isomers. Glycan fucosylation sites were confidently determined by eliminating fucose rearrangement and assignment of diagnostic ions achieved by permethylation and PGC-LC at high temperatures, respectively. Assigning monosaccharide residues to specific glycan antennae was also achieved. Galactose linkages were also distinguished from each other by CID/HCD tandem MS. This was attainable because of the different bond energies associated with monosaccharide linkages.

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“…HILIC is suitable for the analysis of polar analytes which are inadequately retained in RP-LC [1]. HILIC was successfully applied to glycopeptide enrichment [7,8], released glycan analysis [9,10], and separation of different glycoforms of peptides [11]. Although the directed analysis of glycoproteins provides site-specific information on glycans, the common approach to characterize glycans in HILIC is their enzymatic or chemical releas LC/MS analysis [12].…”

Section: Introductionmentioning

confidence: 99%

Nano reversed phase versus nano hydrophilic interaction liquid chromatography on a chip in the analysis of hemopexin glycopeptides

Kozlík

Šanda

Goldman

2017

Journal of Chromatography A

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Analysis of the glycosylation of proteins is a challenge that requires orthogonal methods to achieve separation of the diverse glycoforms. A combination of reversed phase chromatography with tandem mass spectrometry (RP-LC-MS/MS) is one of the most powerful tools for glycopeptide analysis. In this work, we developed and compared RP-LC and hydrophilic interaction liquid chromatography (HILIC) in nanoscale on a chip combined with MS/MS in order to separate glycoforms of two peptides obtained from the tryptic digest of hemopexin. We observed reduction of the retention time with decreasing polarity of glycans attached to the same peptide backbone in HILIC. The opposite effect was observed for RP-LC. The presence of sialic acids prolonged the retention of glycopeptides in both chromatographic modes. The nanoHILIC method provided higher selectivity based on the composition of glycan, compared to nanoRP-LC but a lower sensitivity. The nanoHILIC method was able to partially separate linkage isomers of fucose (core and outer arm) on bi-antennary glycoform of SWPAVGDCSSALR glycopeptide, which is beneficial in the elucidation of the structure of the fucosylated glycoforms.

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HILIC and ERLIC Enrichment of Glycopeptides Derived from Breast and Brain Cancer Cells

Cited by 84 publications

References 36 publications

Study of structure‐dependent chromatographic behavior of glycopeptides using reversed phase nanoLC

Study of structure‐dependent chromatographic behavior of glycopeptides using reversed phase nanoLC

LC-MS/MS analysis of permethylated N-glycans facilitating isomeric characterization

Nano reversed phase versus nano hydrophilic interaction liquid chromatography on a chip in the analysis of hemopexin glycopeptides

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