8 research outputs found
Multiple Enzymatic Digestions and Ion Mobility Separation Improve Quantification of Bacterial Ribosomal Proteins by Data Independent Acquisition Liquid ChromatographyâMass Spectrometry
Mass spectrometry-based quantification
of ribosomal proteins (r-proteins)
associated with mature ribosomes and ribosome assembly complexes is
typically accomplished by relative quantification strategies. These
strategies provide information on the relative stoichiometry of proteins
within the complex compared to a wild-type strain. Here we have evaluated
the applicability of a label-free approach, enhanced liquid chromatographyâmass
spectrometry (LCâMS<sup>E</sup>), for absolute âribosome-centricâ
quantification of r-proteins in Escherichia coli mature ribosomes. Because the information obtained in this experiment
is related to the number of peptides identified per protein, experimental
conditions that allow accurate and reproducible quantification of
r-proteins were found. Using an additional dimension of gas-phase
separation through ion mobility and the use of multiple endoproteinase
digestion significantly improved quantification of proteins associated
with mature ribosomes. The actively translating ribosomes (polysomes)
contain amounts of proteins consistent with their known stoichiometry
within the complex. These measurements exhibited technical and biological
reproducibilities at %CV less than 15% and 35%, respectively. The
improved LCâMS<sup>E</sup> approach described here can be used
to characterize in vivo ribosome assembly complexes captured during
ribosome biogenesis and assembly under different perturbations (e.g.,
antibiotics, deletion mutants of assembly factors, oxidative stress,
nutrient deprivation). Quantitative analysis of these captured complexes
will provide information relating to the interplay and dynamics of
how these perturbations interfere with the assembly process
Targeted Discovery and Validation of Plasma Biomarkers of Parkinsonâs Disease
Despite
extensive research, an unmet need remains for protein biomarkers
of Parkinsonâs disease (PD) in peripheral body fluids, especially
blood, which is easily accessible clinically. The discovery of such
biomarkers is challenging, however, due to the enormous complexity
and huge dynamic range of human blood proteins, which are derived
from nearly all organ systems, with those originating specifically
from the central nervous system (CNS) being exceptionally low in abundance.
In this investigation of a relatively large cohort (âŒ300 subjects),
selected reaction monitoring (SRM) assays (a targeted approach) were
used to probe plasma peptides derived from glycoproteins previously
found to be altered in the CNS based on PD diagnosis or severity.
Next, the detected peptides were interrogated for their diagnostic
sensitivity and specificity as well as the correlation with PD severity,
as determined by the Unified Parkinsonâs Disease Rating Scale
(UPDRS). The results revealed that 12 of the 50 candidate glycopeptides
were reliably and consistently identified in plasma samples, with
three of them displaying significant differences among diagnostic
groups. A combination of four peptides (derived from PRNP, HSPG2,
MEGF8, and NCAM1) provided an overall area under curve (AUC) of 0.753
(sensitivity: 90.4%; specificity: 50.0%). Additionally, combining
two peptides (derived from MEGF8 and ICAM1) yielded significant correlation
with PD severity, that is, UPDRS (<i>r</i> = 0.293, <i>p</i> = 0.004). The significance of these results is at least
two-fold: (1) it is possible to use a targeted approach to identify
otherwise very difficult to detect CNS related biomarkers in peripheral
blood and (2) the novel biomarkers, if validated in independent cohorts,
can be employed to assist with clinical diagnosis of PD as well as
monitoring disease progression