19 research outputs found
Fragmentation of Integral Membrane Proteins in the Gas Phase
Integral membrane proteins (IMPs)
are of great biophysical and
clinical interest because of the key role they play in many cellular
processes. Here, a comprehensive top down study of 152 IMPs and 277
soluble proteins from human H1299 cells including 11 087 fragments
obtained from collisionally activated dissociation (CAD), 6452 from
higher-energy collisional dissociation (HCD), and 2981 from electron
transfer dissociation (ETD) shows their great utility and complementarity
for the identification and characterization of IMPs. A central finding
is that ETD is ∼2-fold more likely to cleave in soluble regions
than threshold fragmentation methods, whereas the reverse is observed
in transmembrane domains with an observed ∼4-fold bias toward
CAD and HCD. The location of charges just prior to dissociation is
consistent with this directed fragmentation: protons remain localized
on basic residues during ETD but easily mobilize along the backbone
during collisional activation. The fragmentation driven by these protons,
which is most often observed in transmembrane domains, both is of
higher yield and occurs over a greater number of backbone cleavage
sites. Further, while threshold dissociation events in transmembrane
domains are on average 10.1 (CAD) and 9.2 (HCD) residues distant from
the nearest charge site (R, K, H, N-terminus), fragmentation is strongly
influenced by the N- or C-terminal position relative to that site:
the ratio of observed b- to y-fragments is ∼1:3 if the cleavage
occurs >7 residues N-terminal and ∼3:1 if it occurs >7
residues
C-terminal to the nearest basic site. Threshold dissociation products
driven by a mobilized proton appear to be strongly dependent on not
only relative position of a charge site but also N- or C-terminal
directionality of proton movement
Comparative top down proteomics of peripheral blood mononuclear cells from kidney transplant recipients with normal kidney biopsies or acute rejection
Recent studies utilizing transcriptomics, metabolomics, and bottom up proteomics have identified molecular signatures of kidney allograft pathology. Although these results make significant progress toward non-invasive differential diagnostics of dysfunction of a transplanted kidney, they provide little information on the intact, often modified, protein molecules present during progression of this pathology. Because intact proteins underpin diverse biological processes, measuring the relative abundance of their modified forms promises to advance mechanistic understanding, and might provide a new class of biomarker candidates. Here, we used top down proteomics to inventory the modified forms of whole proteins in peripheral blood mononuclear cells (PBMCs) taken at the time of kidney biopsy for 40 kidney allograft recipients either with healthy transplants or those suffering acute rejection. Supported by gas-phase fragmentation of whole protein ions during tandem mass spectrometry, we identified 344 proteins mapping to 2,905 distinct molecular forms (proteoforms). Using an initial implementation of a label-free approach to quantitative top down proteomics, we obtained evidence suggesting relative abundance changes in 111 proteoforms between the two patient groups. Collectively, our work is the first to catalog intact protein molecules in PBMCs and suggests differentially abundant proteoforms for further analysis.The following grants are acknowledged: National Institutes of Health P41 GM108569, R01 GM067193 to NLK, P01 AI112522 to NLK and MMA, and U19 A1063603 to DRS
Top Down Proteomics of Human Membrane Proteins from Enriched Mitochondrial Fractions
The interrogation of intact integral membrane proteins
has long
been a challenge for biological mass spectrometry. Here, we demonstrate
the application of top down mass spectrometry to whole membrane proteins
below 60 kDa with up to 8 transmembrane helices. Analysis of enriched
mitochondrial membrane preparations from human cells yielded identification
of 83 integral membrane proteins, along with 163 membrane-associated
or soluble proteins, with a median <i>q</i> value of 3 ×
10<sup>–10</sup>. An analysis of matching fragment ions demonstrated
that significantly more fragment ions were found within transmembrane
domains than would be expected based upon the observed protein sequence.
In total, 46 proteins from the complexes of oxidative phosphorylation
were identified which exemplifies the increasing ability of top down
proteomics to provide extensive coverage in a biological network
Native GELFrEE: A New Separation Technique for Biomolecular Assemblies
The cadre of protein complexes in
cells performs an array of functions
necessary for life. Their varied structures are foundational to their
ability to perform biological functions, lending great import to the
elucidation of complex composition and dynamics. Native separation
techniques that are operative on low sample amounts and provide high
resolution are necessary to gain valuable data on endogenous complexes.
Here, we detail and optimize the use of tube gel separations to produce
samples proven compatible with native, multistage mass spectrometry
(nMS/MS). We find that a continuous system (i.e., no stacking gel)
with a gradient in its extent of cross-linking and use of the clear
native buffer system performs well for both fractionation and native
mass spectrometry of heart extracts and a fungal secretome. This integrated
advance in separations and nMS/MS offers the prospect of untargeted
proteomics at the next hierarchical level of protein organization
in biology
Defining the Neuropeptidome of the Spiny Lobster <i>Panulirus interruptus</i> Brain Using a Multidimensional Mass Spectrometry-Based Platform
Decapod crustaceans are important
animal models for neurobiologists
due to their relatively simple nervous systems with well-defined neural
circuits and extensive neuromodulation by a diverse set of signaling
peptides. However, biochemical characterization of these endogenous
neuropeptides is often challenging due to limited sequence information
about these neuropeptide genes and the encoded preprohormones. By
taking advantage of sequence homology in neuropeptides observed in
related species using a home-built crustacean neuropeptide database,
we developed a semi-automated sequencing strategy to characterize
the neuropeptidome of <i>Panulirus interruptus</i>, an important
aquaculture species, with few known neuropeptide preprohormone sequences.
Our streamlined process searched the high mass accuracy and high-resolution
data acquired on a LTQ-Orbitrap with a flexible algorithm in ProSight
that allows for sequence discrepancy from reported sequences in our
database, resulting in the detection of 32 neuropeptides, including
19 novel ones. We further improved the overall coverage to 51 neuropeptides
with our multidimensional platform that employed multiple analytical
techniques including dimethylation-assisted fragmentation, de novo
sequencing using nanoliquid chromatography-electrospray ionization-quadrupole-time-of-flight
(nanoLC–ESI–Q-TOF), direct tissue analysis, and mass
spectrometry imaging on matrix-assisted laser desorption/ionization
(MALDI)-TOF/TOF. The high discovery rate from this unsequenced model
organism demonstrated the utility of our neuropeptide discovery pipeline
and highlighted the advantage of utilizing multiple sequencing strategies.
Collectively, our study expands the catalog of crustacean neuropeptides
and more importantly presents an approach that can be adapted to exploring
neuropeptidome from species that possess limited sequence information
Robust Analysis of the Yeast Proteome under 50 kDa by Molecular-Mass-Based Fractionation and Top-Down Mass Spectrometry
As the process of top-down mass spectrometry continues to mature, we benchmark the next installment of an improving methodology that incorporates a tube-gel electrophoresis (TGE) device to separate intact proteins by molecular mass. Top-down proteomics is accomplished in a robust fashion to yield the identification of hundreds of unique proteins, many of which correspond to multiple protein forms. The TGE platform separates 0–50 kDa proteins extracted from the yeast proteome into 12 fractions prior to automated nanocapillary LC–MS/MS in technical triplicate. The process may be completed in less than 72 h. From this study, 530 unique proteins and 1103 distinct protein species were identified and characterized, thus representing the highest coverage to date of the Saccharomyces cerevisiae proteome using top-down proteomics. The work signifies a significant step in the maturation of proteomics based on direct measurement and fragmentation of intact proteins
Defining the Neuropeptidome of the Spiny Lobster <i>Panulirus interruptus</i> Brain Using a Multidimensional Mass Spectrometry-Based Platform
Decapod crustaceans are important
animal models for neurobiologists
due to their relatively simple nervous systems with well-defined neural
circuits and extensive neuromodulation by a diverse set of signaling
peptides. However, biochemical characterization of these endogenous
neuropeptides is often challenging due to limited sequence information
about these neuropeptide genes and the encoded preprohormones. By
taking advantage of sequence homology in neuropeptides observed in
related species using a home-built crustacean neuropeptide database,
we developed a semi-automated sequencing strategy to characterize
the neuropeptidome of <i>Panulirus interruptus</i>, an important
aquaculture species, with few known neuropeptide preprohormone sequences.
Our streamlined process searched the high mass accuracy and high-resolution
data acquired on a LTQ-Orbitrap with a flexible algorithm in ProSight
that allows for sequence discrepancy from reported sequences in our
database, resulting in the detection of 32 neuropeptides, including
19 novel ones. We further improved the overall coverage to 51 neuropeptides
with our multidimensional platform that employed multiple analytical
techniques including dimethylation-assisted fragmentation, de novo
sequencing using nanoliquid chromatography-electrospray ionization-quadrupole-time-of-flight
(nanoLC–ESI–Q-TOF), direct tissue analysis, and mass
spectrometry imaging on matrix-assisted laser desorption/ionization
(MALDI)-TOF/TOF. The high discovery rate from this unsequenced model
organism demonstrated the utility of our neuropeptide discovery pipeline
and highlighted the advantage of utilizing multiple sequencing strategies.
Collectively, our study expands the catalog of crustacean neuropeptides
and more importantly presents an approach that can be adapted to exploring
neuropeptidome from species that possess limited sequence information
Robust Analysis of the Yeast Proteome under 50 kDa by Molecular-Mass-Based Fractionation and Top-Down Mass Spectrometry
As the process of top-down mass spectrometry continues to mature, we benchmark the next installment of an improving methodology that incorporates a tube-gel electrophoresis (TGE) device to separate intact proteins by molecular mass. Top-down proteomics is accomplished in a robust fashion to yield the identification of hundreds of unique proteins, many of which correspond to multiple protein forms. The TGE platform separates 0–50 kDa proteins extracted from the yeast proteome into 12 fractions prior to automated nanocapillary LC–MS/MS in technical triplicate. The process may be completed in less than 72 h. From this study, 530 unique proteins and 1103 distinct protein species were identified and characterized, thus representing the highest coverage to date of the Saccharomyces cerevisiae proteome using top-down proteomics. The work signifies a significant step in the maturation of proteomics based on direct measurement and fragmentation of intact proteins