61 research outputs found
Higher Temperature Porous Graphitic Carbon Separations Differentially Impact Distinct Glycopeptide Classes
Mass spectrometry-based
discovery glycoproteomics is
highly dependent
on the use of chromatography paradigms amenable to analyte retention
and separation. When compared against established stationary phases
such as reversed-phase and hydrophilic interaction liquid chromatography,
reports utilizing porous graphitic carbon have detailed its numerous
advantages. Recent efforts have highlighted the utility in porous
graphitic carbon in high-throughput glycoproteomics, principally through
enhanced profiling depth and liquid-phase resolution at higher column
temperatures. However, increasing column temperature has been shown
to impart disparaging effects in glycopeptide identification. Herein
we further elucidate this trend, describing qualitative and semiquantitative
effects of increased column temperature on glycopeptide identification
rates, signal intensity, resolution, and spectral count linear response.
Through analysis of enriched bovine and human glycopeptides, species
with high mannose and sialylated glycans were shown to most significantly
benefit and suffer from high column temperatures, respectively. These
results provide insight as to how porous graphitic carbon separations
may be appropriately leveraged for glycopeptide identification while
raising concerns over quantitative and semiquantitative label-free
comparisons as the temperature changes. RAW MS glycoproteomic data
are available via ProteomeXchange with identifier PXD034354
Inclusion of Porous Graphitic Carbon Chromatography Yields Greater Protein Identification and Compartment and Process Coverage and Enables More Reflective Protein-Level Label-Free Quantitation
The ubiquity of mass spectrometry-based
bottom-up proteomic analyses
as a component of biological investigation mandates the validation
of methodologies that increase acquisition efficiency, improve sample
coverage, and enhance profiling depth. Chromatographic separation
is often ignored as an area of potential improvement, with most analyses
relying on traditional reversed-phase liquid chromatography (RPLC);
this consistent reliance on a single chromatographic paradigm fundamentally
limits our view of the observable proteome. Herein, we build upon
early reports and validate porous graphitic carbon chromatography
(PGC) as a facile means to substantially enhance proteomic coverage
without changes to sample preparation, instrument configuration, or
acquisition methods. Analysis of offline fractionated cell line digests
using both separations revealed an increase in peptide and protein
identifications by 43% and 24%, respectively. Increased identifications
provided more comprehensive coverage of cellular components and biological
processes independent of protein abundance, highlighting the substantial
quantity of proteomic information that may go undetected in standard
analyses. We further utilize these data to reveal that label-free
quantitative analyses using RPLC separations alone may not be reflective
of actual protein constituency. Together, these data highlight the
value and comprehension offered through PGC-MS proteomic analyses.
RAW proteomic data have been uploaded to the MassIVE repository with
the primary accession code MSV000091495
Higher Temperature Porous Graphitic Carbon Separations Differentially Impact Distinct Glycopeptide Classes
Mass spectrometry-based
discovery glycoproteomics is
highly dependent
on the use of chromatography paradigms amenable to analyte retention
and separation. When compared against established stationary phases
such as reversed-phase and hydrophilic interaction liquid chromatography,
reports utilizing porous graphitic carbon have detailed its numerous
advantages. Recent efforts have highlighted the utility in porous
graphitic carbon in high-throughput glycoproteomics, principally through
enhanced profiling depth and liquid-phase resolution at higher column
temperatures. However, increasing column temperature has been shown
to impart disparaging effects in glycopeptide identification. Herein
we further elucidate this trend, describing qualitative and semiquantitative
effects of increased column temperature on glycopeptide identification
rates, signal intensity, resolution, and spectral count linear response.
Through analysis of enriched bovine and human glycopeptides, species
with high mannose and sialylated glycans were shown to most significantly
benefit and suffer from high column temperatures, respectively. These
results provide insight as to how porous graphitic carbon separations
may be appropriately leveraged for glycopeptide identification while
raising concerns over quantitative and semiquantitative label-free
comparisons as the temperature changes. RAW MS glycoproteomic data
are available via ProteomeXchange with identifier PXD034354
- …