Ultrasensitive Characterization of Site-Specific Glycosylation of Affinity-Purified Haptoglobin from Lung Cancer Patient Plasma Using 10 μm i.d. Porous Layer Open Tubular Liquid Chromatography−Linear Ion Trap Collision-Induced Dissociation/Electron Transfer Dissociation Mass Spectrometry

Abstract

Site-specific analysis of protein glycosylation is important for biochemical and clinical research efforts. Glycopeptide analysis using liquid chromatography -collision induced dissociation/ electron transfer dissociation -mass spectrometry (LC-CID/ETD-MS) allows simultaneous characterization of glycan structure and attached peptide site. However, due to the low ionization efficiency of glycopeptides during electrospray ionization (ESI), 200-500 fmol of sample per injection is needed for a single LC-MS run, which makes it challenging for the analysis of limited amounts of glycoprotein purified from biological matrices. To improve the sensitivity of LC-MS analysis for glycopeptides, an ultra-narrow porous layer open tubular (PLOT) LC column (2.5 m × 10 μm i.d.) was coupled to a linear ion trap mass spectrometer (LTQ-CID/ETD-MS) to provide sensitive analysis of N-linked protein glycosylation heterogeneity. The potential of the developed method is demonstrated by the characterization of site-specific glycosylation using haptoglobin (Hpt) as a model protein. To limit the amount of haptoglobin to low pmole amounts of protein, we affinity purified it from 1 μL of pooled lung cancer patients plasma. A total of 26 glycoforms/ glycan compositions on three Hpt tryptic glycopeptides were identified and quantified from 10 LC-MS runs with a consumption of 100 fmol Hpt digest (13 ng protein, 10 fmol per injection). Included in this analysis was the determination of the glycan occupancy level. At this sample consumption level, the high sensitivity of the PLOT LC-LTQ-CID/ETD-MS allowed glycopeptide identification and structure determination, along with relative quantitation of glycans presented on the same peptide backbone, even for low abundant glycopeptides at the ~100 attomole level. The PLOT LC-MS is shown to have sufficient sensitivity to allow characterization of site-specific protein glycosylation from trace levels of glycosylated proteins.

An Open Tubular CEC Column of Excellent Separation Efficiency for Proteomic Analysis

“…Such LC column format has been actually developed and employed in proteomic analysis mostly coupled to mass spectrometry in gradient elution mode. [19][20][21][22][23][24] Long and narrow silica capillaries of ca 10 µm I.D. and 3-5 m length have been modified to have a thin (ca 1-2 µm) porous layer open tubular (PLOT) polymer film on the inner surface of capillary, and the resultant columns showed superb peak separation capacity.…”

“…and 3-5 m length have been modified to have a thin (ca 1-2 µm) porous layer open tubular (PLOT) polymer film on the inner surface of capillary, and the resultant columns showed superb peak separation capacity. [19][20][21][22][23][24] Such narrow I.D. was required in LC application to minimize band broadening.…”

Energy-resolved collision-induced dissociation pathways of model N-linked glycopeptides: implications for capturing glycan connectivity and peptide sequence in a single experiment

“…[34][35][36][37][38] This general behavior has proven very valuable, as application of two complementary methods can enable thorough characterization of glycopeptide composition and structure. [39][40][41][42][43] Unsurprisingly, a number of other glycopeptide ion dissociation pathways have been observed which do not fall within the domain of strictly complementarity vibrational activation/dissociation and electron transfer/ capture dissociation. [44][45][46][47] This includes numerous examples of vibrational activation/dissociation methods providing information not limited to the carbohydrate group, but extending to the amino acid sequence of the peptide group as well.…”

Veterinary Biomarker Discovery: Proteomic Analysis of Acute Phase Proteins

“…The fragmentation of the peptide backbone using ETD allows for amino acid side chains and modifications such as glycosylation and phosphorylation to remain intact, making it possible not only to deduce the amino acid sequence of a peptide, but also to detect any modified residues (Syka et al, 2004). Furthermore, the combination of CID and ETD has proven effective as well in the characterization of isolated glycopeptides, including modified peptides from HPT in human lung cancer patients (Wang et al, 2011).…”