Deborah Leon | Boston University (original) (raw)

Papers by Deborah Leon

Proteomic tools identify constituents of complex mixtures, often delivering long lists of identi... more Proteomic tools identify constituents of complex mixtures, often delivering long lists
of identified proteins. The high-throughput methods excel at matching tandem mass
spectrometry data to spectra predicted from sequence databases. Unassigned mass
spectra are ignored, but could, in principle, provide valuable information on unanticipated
modifications and improve protein annotations while consuming limited quantities of
material. Strategies to “mine” information from these discards are presented, along with
discussion of features that, when present, provide strong support for modifications. In
this study we mined LC-MS/MS datasets of proteolytically-digested concanavalin A pull
down fractions from Methanosarcina mazei Gö1 cell lysates. Analyses identified 154
proteins. Many of the observed proteins displayed post-translationally modified forms,
including O-formylated and methyl-esterified segments that appear biologically relevant
(i.e., not artifacts of sample handling). Interesting cleavages and modifications (e.g.,
S-cyanylation and trimethylation) were observed near catalytic sites of methanogenesis
enzymes. Of 31 Methanosarcina protein N-termini recovered by concanavalin A binding or
from a previous study, only M. mazei S-layer protein MM1976 and its M. acetivorans C2A
orthologue, MA0829, underwent signal peptide excision. Experimental results contrast
with predictions from algorithms SignalP 3.0 and Exprot, which were found to over-predict
the presence of signal peptides. Proteins MM0002, MM0716, MM1364, and MM1976
were found to be glycosylated, and employing chromatography tailored specifically
for glycopeptides will likely reveal more. This study supplements limited, existing
experimental datasets of mature archaeal N-termini, including presence or absence of
signal peptides, translation initiation sites, and other processing. Methanosarcina surface
and membrane proteins are richly modified.

Extracellular matrixes comprise glycoproteins, glycosaminoglycans and proteoglycans that order th... more Extracellular matrixes comprise glycoproteins, glycosaminoglycans and proteoglycans that order the environment through which cells receive signals and communicate. Proteomic and glycomic molecular signatures from tissue surfaces can add diagnostic power to the immunohistochemistry workflows. Acquired in a spatially resolved manner, such proteomic and glycomic information can help characterize disease processes and be easily applied in a clinical setting. Our aim toward obtaining integrated omics datasets was to develop the first workflow applicable for simultaneous analysis of glycosaminoglycans, N-glycans and proteins/peptides from tissue surface areas as small as 1.5 mm in diameter. Targeting small areas is especially important in the case of glycans, as their distribution can be very heterogeneous between different tissue regions. We first established reliable and reproducible digestion protocols for the individual compound classes by applying standards on the tissue using microwave irradiation to achieve reduced digestion times. Next, we developed a multienzyme workflow suitable for analysis of the different compound classes. Applicability of the workflow was demonstrated on serial mouse brain and liver sections, both fresh frozen and formalin-fixed. The glycomics data from the 1.5 mm diameter tissue surface area was consistent with data published on bulk mouse liver and brain tissues, which demonstrates the power of the workflow in obtaining combined molecular signatures from very small tissue regions.

A glycoprotein may contain several sites of glycosylation, each of which is heterogeneous. As a c... more A glycoprotein may contain several sites of glycosylation, each of which is heterogeneous. As a consequence of glycoform diversity and signal suppression from nonglycosylated peptides that ionize more efficiently, typical reversed-phase LC-MS and bottom-up proteomics database searching workflows do not perform well for identification of site-specific glycosylation for complex glycoproteins. We present an LC-MS system for enrichment, separation, and analysis of glycopeptides from complex glycoproteins (>4 N-glycosylation sequons) in a single step. This system uses an online HILIC enrichment trap prior to reversed-phase C18-MS analysis. We demonstrated the effectiveness of the system using a set of glycoproteins including human transferrin (2 sequons), human alpha-1-acid glycoprotein (5 sequons), and influenza A virus hemagglutinin (9 sequons). The online enrichment renders glycopeptides the most abundant ions detected, thereby facilitating the generation of high-quality data-dependent tandem mass spectra. The tandem mass spectra exhibited product ions from both glycan and peptide backbone dissociation for a majority of the glycopeptides tested using collisionally activated dissociation that served to confidently assign site-specific glycosylation. We demonstrated the value of our system to define site-specific glycosylation using a hemagglutinin containing 9 N-glycosylation sequons from a single HILIC-C18-MS acquisition.

Many archaeal cell envelopes contain a protein coat or sheath composed of one or more surface exp... more Many archaeal cell envelopes contain a protein coat or sheath composed of one or more surface exposed proteins. These surface layer (S-layer) proteins contribute structural integrity and protect the lipid membrane from environmental challenges. To explore the species diversity of these layers in the Methanosarcinaceae, the major S-layer protein in Methanosarcina barkeri strain Fusaro was identified using proteomics. The Mbar_A1758 gene product was present in multiple forms with apparent sizes of 130, 120, and 100 kDa, consistent with post-translational modifications including signal peptide excision and protein glycosylation. A protein with features related to the surface layer proteins found in Methanosarcina acetivorans C2A and Methanosarcina mazei Goel was identified in the M. barkeri genome. These data reveal a distinct conserved protein signature with features and implied cell surface architecture in the Methanosarcinaceae that is absent in other archaea. Paralogous gene expression patterns in two Methanosarcina species revealed abundant expression of a single S-layer paralog in each strain. Respective promoter elements were identified and shown to be conserved in mRNA coding and upstream untranslated regions. Prior M. acetivorans genome annotations assigned S-layer or surface layer associated roles of eighty genes: however, of 68 examined none was significantly expressed relative to the experimentally determined S-layer gene.

The outermost cell envelope structure of many archaea and bacteria contains a proteinaceous latti... more The outermost cell envelope structure of many archaea and bacteria contains a proteinaceous lattice termed the surface layer or S-layer. It is typically composed of only one or two abundant, often posttranslationally modified proteins that self-assemble to form the highly organized arrays. Surprisingly, over 100 proteins were annotated to be S-layer components in the archaeal species Methanosarcina acetivorans C2A and Methanosarcina mazei Gö1, reflecting limitations of current predictions. An in vivo biotinylation methodology was devised to affinity tag surface-exposed proteins while overcoming unique challenges in working with these fragile organisms. Cells were adapted to growth under N2 fixing conditions, thus, minimizing free amines reactive to the NHS-label, and high pH media compatible with the acylation chemistry was used. A 3-phase separation procedure was employed to isolate intact, labeled cells from lysed-cell derived proteins. Streptavidin affinity enrichment followed by stringent wash conditions removed nonspecifically bound proteins. This methodology revealed S-layer proteins in M. acetivorans C2A and M. mazei Gö1 to be MA0829 and MM1976, respectively. Each was demonstrated to exist as multiple glycosylated forms using SDS-PAGE coupled with glycoprotein-specific staining, and by interaction with the lectin, Concanavalin A. A number of additional surface-exposed proteins and glycoproteins were identified and included all three subunits of the thermosome: the latter suggests that the chaperonin complex is both surface- and cytoplasmically localized. This approach provides an alternative strategy to study surface proteins in the archaea.