Glycosylation analyses were performed by quantitation of intact transferrin isoforms through isoelectric focusing (IEF) and/or capillary zone electrophoresis (CZE) using the carbohydrate-deficient transferrin (CDT) test. Transferrin isoforms percentage was compared with internal reference values. In-depth transferrin and total serum N-glycan structural analyses were conducted by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). In this case, transferrin was isolated from serum by immunoaffinity depletion on IgY microbead spin columns (Seppro™ GenWay Biotech, San Diego, CA) and deglycosylated using PNGase F [8 (link)]. Transferrin N-glycans from the patient and age-matched controls’ sera were analyzed after sample permethylation [9 (link)]. Total N-glycan profiling was obtained by using 10 μL of serum as previously described [8 (link)]. Briefly, proteins were denatured with RapiGest™ SF surfactant, reduced and alkylated with DTT and IAA respectively, then deglycosylated by PNGase F. Released N-glycans were permethylated prior to MALDI-TOF MS analysis, performed in reflector mode and in positive polarity on a 4800 Proteomic Analyzer (AB Sciex). Total N-glycans spectra from age-matched control sera were used for comparison.
4800 proteomic analyzer
Manufactured by AB Sciex
Sourced in United States
The 4800 Proteomic Analyzer is a mass spectrometry instrument designed for protein analysis. It is capable of high-throughput, automated protein identification and characterization. The core function of the 4800 Proteomic Analyzer is to provide accurate mass measurements and fragmentation data for protein samples.
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Lab products found in correlation
5 protocols using 4800 proteomic analyzer
1
Glycosylation Profiling by Mass Spectrometry
2
MALDI-TOF Mass Spectrometry of LPS
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MALDI-TOF mass spectrometry of purified LPS samples was performed on a 4800 Proteomic Analyzer (ABSciex, USA), as described (Sturiale et al., 2011 (link)). Negative ion mass spectra were acquired in reflector modes with mass accuracy ca. 50 ppm. 2′,4′,6′-Trihydroxyacetophenone monohydrate was used for matrix preparation. Mass spectra were analyzed as described (Sturiale et al., 2011 (link)).
3
MALDI-TOF Mass Spectrometry of Purified LPS
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MALDI-TOF mass spectrometry of purified LPS samples was performed on a Voyager STR system (PerSeptive, Framingham, MA, USA) and a 4800 Proteomic Analyzer (ABSciex, USA), as described (33 (link)). Negative ion mass spectra were acquired in both linear and reflector modes with mass accuracy ca. 50 ppm. 2′,4′,6′-Trihydroxyacetophenone monohydrate was used for matrix preparation. Mass spectra were analyzed as described (33 (link)).
4
MALDI-TOF Mass Spectrometry of Intact LOS
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A 4800 Proteomic Analyzer (AB Sciex) was used, equipped with a Nd:YAG laser operating at a wavelength of 355 nm with a <500 ps pulse and 200 Hz firing rate. MALDI‐TOF mass spectra of intact LOS were recorded either in linear or reflectron mode using positive‐ion polarity. Each spectrum resulted from the sum of around 2000 laser shots. Mass spectra acquired in the reflector mode allowed detection of monoisotopic masses. External calibration using a dedicated peptide calibration mixture (AB Sciex), provided mass accuracy below 75 ppm. Data were processed using DataExplorer 4.9 software. The MS/MS experiments reported in this study were performed without use of a collision gas. Native LOS samples were prepared as previously reported.34
5
MALDI-TOF MS Analysis of Permethylated N-Glycans
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MALDI-TOF MS data were acquired in positive polarity on a 4800 Proteomic Analyzer (AB Sciex) equipped with a Nd:YAG laser operating at a wavelength of 355 nm with <500-ps pulse and 200-Hz firing rate. Each spectrum was the result of the sum of about 2000 laser shots. Mass spectra of permethylated N-glycans were acquired in reflector mode allowing the detection of monoisotopic masses. An external calibration using a dedicated peptide calmix (AB Sciex) provided mass accuracy below 45 ppm. Data were processed using Data Explorer® software 4.9.
Structural assignments were based on molecular weight identifications, knowledge of the N-glycan biosynthetic pathway. N-glycan species were identified by using bioinformatic tools, such as GlycoMod (http://web.expasy.org/glycomod/), Glycoworkbench v2.1 [28] and by tools provided by the consortium for functional glycomics (CFG; http://www. functionalglycomics.org). All the glycomic data were shown as Supplementary Table 1. Raw files were uploaded to Glycopost (https://glycopost.glycosmos.org/).
Structural assignments were based on molecular weight identifications, knowledge of the N-glycan biosynthetic pathway. N-glycan species were identified by using bioinformatic tools, such as GlycoMod (http://web.expasy.org/glycomod/), Glycoworkbench v2.1 [28] and by tools provided by the consortium for functional glycomics (CFG; http://www. functionalglycomics.org). All the glycomic data were shown as Supplementary Table 1. Raw files were uploaded to Glycopost (https://glycopost.glycosmos.org/).
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