3 research outputs found
Quantitative Structural Characterization of Local N‑Glycan Microheterogeneity in Therapeutic Antibodies by Energy-Resolved Oxonium Ion Monitoring
Site-specific characterization of glycoform heterogeneity
currently
requires glycan structure assignment and glycopeptide quantification
in two independent experiments. We present here a new method combining
multiple reaction monitoring mass spectrometry with energy-resolved
structural analysis, which we termed “energy-resolved oxonium
ion monitoring”. We demonstrated that monitoring the yields
of oligosaccharide-derived fragment ions (oxonium ions) over a wide
range of collision induced dissociation (CID) energy applied to a
glycopeptide precursor exhibits a glycan structure-unique fragmentation
pattern. In the analysis of purified immunoglobulin glycopeptides,
the energy-resolved oxonium ion profile was shown to clearly distinguish
between isomeric glycopeptides. Moreover, limit of detection (LOD)
of glycopeptide detection was 30 attomole injection, and quantitative
dynamic range spanned 4 orders magnitude. Therefore, both quantification
of glycopeptides and assignment of their glycan structures were achieved
by a simple analysis procedure. We assessed the utility of this method
for characterizing site-specific N-glycan microheterogeneity on therapeutic
antibodies, including validation of lot-to-lot glycoform variability.
A significant change in the degree of terminal galactosylation was
observed in different production lots of trastuzumab and bevacizumab.
Cetuximab Fab glycosylation, previously known to cause anaphylaxis,
was also analyzed, and several causative antigens including Lewis
X motifs were quantitatively detected. The data suggests that energy-resolved
oxonium ion monitoring could fulfill the regulatory requirement on
the routine quality control analysis of forthcoming biosimilar therapeutics
Plasma Low-Molecular-Weight Proteome Profiling Identified Neuropeptide‑Y as a Prostate Cancer Biomarker Polypeptide
In
prostate cancer diagnosis, PSA test has greatly contributed
to the early detection of prostate cancer; however, expanding overdiagnosis
and unnecessary biopsies have emerged as serious issues. To explore
plasma biomarkers complementing the specificity of PSA test, we developed
a unique proteomic technology QUEST-MS (Quick Enrichment of Small
Targets for Mass Spectrometry). The QUEST-MS method based on 96-well
formatted sequential reversed-phase chromatography allowing efficient
enrichment of <20 kDa proteins quickly and reproducibly. Plasma
from 24 healthy controls, 19 benign prostate hypertrophy patients,
and 73 prostate cancer patients were purified with QUEST-MS and analyzed
by LC/MS/MS. Among 153 057 nonredundant peptides, 189 peptides showed
prostate cancer specific detection pattern, which included a neurotransmitter
polypeptide neuropeptide-Y (NPY). We further validated the screening
results by targeted multiple reaction monitoring technology using
independent sample set (<i>n</i> = 110). The ROC curve analysis
revealed that logistic regression-based combination of NPY, and PSA
showed 81.5% sensitivity and 82.2% specificity for prostate cancer
diagnosis. Thus QUEST-MS technology allowed comprehensive and high-throughput
profiling of plasma polypeptides and had potential to effectively
uncover very low abundant tumor-derived small molecules, such as neurotransmitters,
peptide hormones, or cytokines
Plasma Low-Molecular-Weight Proteome Profiling Identified Neuropeptide‑Y as a Prostate Cancer Biomarker Polypeptide
In
prostate cancer diagnosis, PSA test has greatly contributed
to the early detection of prostate cancer; however, expanding overdiagnosis
and unnecessary biopsies have emerged as serious issues. To explore
plasma biomarkers complementing the specificity of PSA test, we developed
a unique proteomic technology QUEST-MS (Quick Enrichment of Small
Targets for Mass Spectrometry). The QUEST-MS method based on 96-well
formatted sequential reversed-phase chromatography allowing efficient
enrichment of <20 kDa proteins quickly and reproducibly. Plasma
from 24 healthy controls, 19 benign prostate hypertrophy patients,
and 73 prostate cancer patients were purified with QUEST-MS and analyzed
by LC/MS/MS. Among 153 057 nonredundant peptides, 189 peptides showed
prostate cancer specific detection pattern, which included a neurotransmitter
polypeptide neuropeptide-Y (NPY). We further validated the screening
results by targeted multiple reaction monitoring technology using
independent sample set (<i>n</i> = 110). The ROC curve analysis
revealed that logistic regression-based combination of NPY, and PSA
showed 81.5% sensitivity and 82.2% specificity for prostate cancer
diagnosis. Thus QUEST-MS technology allowed comprehensive and high-throughput
profiling of plasma polypeptides and had potential to effectively
uncover very low abundant tumor-derived small molecules, such as neurotransmitters,
peptide hormones, or cytokines