12 research outputs found
Deglycosylation and label-free quantitative LC-MALDI MS applied to efficient serum biomarker discovery of lung cancer
<p>Abstract</p> <p>Background</p> <p>Serum is an ideal source of biomarker discovery and proteomic profiling studies are continuously pursued on serum samples. However, serum is featured by high level of protein glycosylations that often cause ionization suppression and confound accurate quantification analysis by mass spectrometry. Here we investigated the effect of N-glycan and sialic acid removal from serum proteins on the performance of label-free quantification results.</p> <p>Results</p> <p>Serum tryptic digests with or without deglycosylation treatment were analyzed by LC-MALDI MS and quantitatively compared on the Expressionist Refiner MS module. As a result, 345 out of 2,984 peaks (11.6%) showed the specific detection or the significantly improved intensities in deglycosylated serum samples (<it>P </it>< 0.01). We then applied this deglycosylation-based sample preparation to the identification of lung cancer biomarkers. In comparison between 10 healthy controls and 20 lung cancer patients, 40 peptides were identified to be differentially presented (<it>P </it>< 0.01). Their quantitative accuracies were further verified by multiple reaction monitoring. The result showed that deglycosylation was needed for the identification of some unique candidates, including previously unreported O-linked glycopeptide of complement component C9.</p> <p>Conclusions</p> <p>We demonstrated here that sample deglycosylation improves the quantitative performance of shotgun proteomics, which can be effectively applied to any samples with high glycoprotein contents.</p
A Comprehensive Peptidome Profiling Technology for the Identification of Early Detection Biomarkers for Lung Adenocarcinoma
The mass spectrometry-based peptidomics approaches have proven its usefulness in several areas such as the discovery of physiologically active peptides or biomarker candidates derived from various biological fluids including blood and cerebrospinal fluid. However, to identify biomarkers that are reproducible and clinically applicable, development of a novel technology, which enables rapid, sensitive, and quantitative analysis using hundreds of clinical specimens, has been eagerly awaited. Here we report an integrative peptidomic approach for identification of lung cancer-specific serum peptide biomarkers. It is based on the one-step effective enrichment of peptidome fractions (molecular weight of 1,000–5,000) with size exclusion chromatography in combination with the precise label-free quantification analysis of nano-LC/MS/MS data set using Expressionist proteome server platform. We applied this method to 92 serum samples well-managed with our SOP (standard operating procedure) (30 healthy controls and 62 lung adenocarcinoma patients), and quantitatively assessed the detected 3,537 peptide signals. Among them, 118 peptides showed significantly altered serum levels between the control and lung cancer groups (p<0.01 and fold change >5.0). Subsequently we identified peptide sequences by MS/MS analysis and further assessed the reproducibility of Expressionist-based quantification results and their diagnostic powers by MRM-based relative-quantification analysis for 96 independently prepared serum samples and found that APOA4 273–283, FIBA 5–16, and LBN 306–313 should be clinically useful biomarkers for both early detection and tumor staging of lung cancer. Our peptidome profiling technology can provide simple, high-throughput, and reliable quantification of a large number of clinical samples, which is applicable for diverse peptidome-targeting biomarker discoveries using any types of biological specimens
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
The uptake and the transport of14C-labeled epibrassinolide in intact seedlings of cucumber and wheat
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