6 research outputs found
In-Gel Nonspecific Proteolysis for Elucidating Glycoproteins: A Method for Targeted Protein-Specific Glycosylation Analysis in Complex Protein Mixtures
Determining protein-specific glycosylation in protein
mixtures
remains a difficult task. A common approach is to use gel electrophoresis
to isolate the protein followed by glycan release from the identified
band. However, gel bands are often composed of several proteins. Hence,
release of glycans from specific bands often yields products not from
a single protein but a composite. As an alternative, we present an
approach whereby glycans are released with peptide tags allowing verification
of glycans bound to specific proteins. We term the process in-gel
nonspecific proteolysis for elucidating glycoproteins (INPEG). INPEG
combines rapid gel separation of a protein mixture with in-gel nonspecific
proteolysis of protein bands followed by tandem mass spectrometry
(MS) analysis of the resulting N- and O-glycopeptides. Here, in-gel
digestion is shown for the first time with nonspecific and broad specific
proteases such as Pronase, proteinase K, pepsin, papain, and subtilisin.
Tandem MS analysis of the resulting glycopeptides separated on a porous
graphitized carbon (PGC) chip was achieved via nanoflow liquid chromatography
coupled with quadrupole time-of-flight mass spectrometry (nano-LC/Q-TOF
MS). In this study, rapid and automated glycopeptide assignment was
achieved via an in-house software (Glycopeptide Finder) based on a
combination of accurate mass measurement, tandem MS data, and predetermined
protein identification (obtained via routine shotgun analysis). INPEG
is here initially validated for O-glycosylation (Îş casein) and
N-glycosylation (ribonuclease B). Applications of INPEG were further
demonstrated for the rapid determination of detailed site-specific
glycosylation of lactoferrin and transferrin following gel separation
and INPEG analysis on crude bovine milk and human serum, respectively
Isomer-Specific LC/MS and LC/MS/MS Profiling of the Mouse Serum N‑Glycome Revealing a Number of Novel Sialylated N‑Glycans
Mice
are the premier mammalian models for studies of human physiology
and disease, bearing extensive biological similarity to humans with
far fewer ethical, economic, or logistic complications. To facilitate
glycomic studies based on the mouse model, we comprehensively profiled
the mouse serum N-glycome using isomer-specific nano-LC/MS and -LC/MS/MS.
N-Glycans were identified by accurate mass MS and structurally elucidated
by MS/MS. Porous graphitized carbon nano-LC was able to separate out
nearly 300 N-linked glycan compounds (including isomers) from just
over 100 distinct N-linked glycan compositions. Additional MS/MS structural
analysis was performed on a number of novel N-glycans, revealing the
structural characteristics of modifications such as dehydration, O-acetylation,
and lactylation. Experimental findings were combined with known glycobiology
to generate a theoretical library of all biologically possible mouse
serum N-glycan compositions. The library may be used for automated
identification of complex mixtures of mouse N-glycans, with possible
applications to a wide range of mouse-related research endeavors,
including pharmaceutical drug development and biomarker discovery
Isomer-Specific LC/MS and LC/MS/MS Profiling of the Mouse Serum N‑Glycome Revealing a Number of Novel Sialylated N‑Glycans
Mice
are the premier mammalian models for studies of human physiology
and disease, bearing extensive biological similarity to humans with
far fewer ethical, economic, or logistic complications. To facilitate
glycomic studies based on the mouse model, we comprehensively profiled
the mouse serum N-glycome using isomer-specific nano-LC/MS and -LC/MS/MS.
N-Glycans were identified by accurate mass MS and structurally elucidated
by MS/MS. Porous graphitized carbon nano-LC was able to separate out
nearly 300 N-linked glycan compounds (including isomers) from just
over 100 distinct N-linked glycan compositions. Additional MS/MS structural
analysis was performed on a number of novel N-glycans, revealing the
structural characteristics of modifications such as dehydration, O-acetylation,
and lactylation. Experimental findings were combined with known glycobiology
to generate a theoretical library of all biologically possible mouse
serum N-glycan compositions. The library may be used for automated
identification of complex mixtures of mouse N-glycans, with possible
applications to a wide range of mouse-related research endeavors,
including pharmaceutical drug development and biomarker discovery
Automated Assignments of N- and O‑Site Specific Glycosylation with Extensive Glycan Heterogeneity of Glycoprotein Mixtures
Site-specific glycosylation (SSG)
of glycoproteins remains a considerable
challenge and limits further progress in the areas of proteomics and
glycomics. Effective methods require new approaches in sample preparation,
detection, and data analysis. While the field has advanced in sample
preparation and detection, automated data analysis remains an important
goal. A new bioinformatics approach implemented in software called
GP Finder automatically distinguishes correct assignments from random
matches and complements experimental techniques that are optimal for
glycopeptides, including nonspecific proteolysis and high mass resolution
liquid chromatography/tandem mass spectrometry (LC/MS/MS). SSG for
multiple N- and O-glycosylation sites, including extensive glycan
heterogeneity, was annotated for single proteins and protein mixtures
with a 5% false-discovery rate, generating hundreds of nonrandom glycopeptide
matches and demonstrating the proof-of-concept for a self-consistency
scoring algorithm shown to be compliant with the target-decoy approach
(TDA). The approach was further applied to a mixture of N-glycoproteins
from unprocessed human milk and O-glycoproteins from very-low-density-lipoprotein
(vLDL) particles
Spatially-Resolved Exploration of the Mouse Brain Glycome by Tissue Glyco-Capture (TGC) and Nano-LC/MS
Tissue glyco-capture (TGC), a highly
sensitive MS-compatible method
for extraction of glycans from tissue, was combined with structure-specific
nano-LC/MS for sensitive and detailed profiling of the mouse brain
glycome. Hundreds of glycan structures were directly detected by accurate
mass MS and structurally elucidated by MS/MS, revealing the presence
of novel glycan motifs such as antennary fucosylation, sulfation,
and glucuronidation that are potentially associated with cellular
signaling and adhesion. Microgram-level sensitivity enabled glycomic
analysis of specific regions of the brain, as demonstrated on not
only brain sections (with a one-dimensional spatial resolution of
20 ÎĽm) but also isolated brain structures (e.g., the hippocampus).
Reproducibility was extraordinarily high (<i>R</i> >
0.98)
for both method and instrumental replicates. The pairing of TGC with
structure-specific nano-LC/MS was found to be an exceptionally powerful
platform for qualitative and quantitative exploration of the brain
glycome
Differentiation of Cancer Cell Origin and Molecular Subtype by Plasma Membrane N‑Glycan Profiling
In clinical settings,
biopsies are routinely used to determine
cancer type and grade based on tumor cell morphology, as determined
via histochemical or immunohistochemical staining. Unfortunately,
in a significant number of cases, traditional biopsy results are either
inconclusive or do not provide full subtype differentiation, possibly
leading to inefficient or ineffective treatment. Glycomic profiling
of the cell membrane offers an alternate route toward cancer diagnosis.
In this study, isomer-sensitive nano-LC/MS was used to directly obtain
detailed profiles of the different N-glycan structures present on
cancer cell membranes. Membrane N-glycans were extracted from cells
representing various subtypes of breast, lung, cervical, ovarian,
and lymphatic cancer. Chip-based porous graphitized carbon nano-LC/MS
was used to separate, identify, and quantify the native N-glycans.
Structure-sensitive N-glycan profiling identified hundreds of glycan
peaks per cell line, including multiple isomers for most compositions.
Hierarchical clusterings based on Pearson correlation coefficients
were used to quickly compare and separate each cell line according
to originating organ and disease subtype. Based simply on the relative
abundances of broad glycan classes (e.g., high mannose, complex/hybrid
fucosylated, complex/hybrid sialylated, etc.), most cell lines were
readily differentiated. More closely related cell lines were differentiated
based on several-fold differences in the abundances of individual
glycans. Based on characteristic N-glycan profiles, primary cancer
origins and molecular subtypes could be distinguished. These results
demonstrate that stark differences in cancer cell membrane glycosylation
can be exploited to create an MS-based biopsy, with potential applications
toward cancer diagnosis and direction of treatment