Biology subjects

McGee, J. P.

Publications and source records attributed to McGee, J. P..

7 recordsLinked to original sources

bioRxiv2026

Deep Proteoform Sequencing with Top-Down Direct Mass Technology

Individual Ion Mass Spectrometry (I2MS) using Direct Mass Technology mode on an Orbitrap mass spectrometer (DMTm) increases sensitivity, resolution, and mass range for protein analysis. Here, we present an end-to-end workflow for deep proteoform sequencing using top-down mass spectrometry with DMTm. By assigning the charge of individual fragment ions and converting spectra from the m/z to the mass domain, DMTm resolves overlapping isotopic distributions that have limited conventional top-down mass spectrometry. Across different fragmentation modes on Orbitrap mass spectrometers, top-down DMTm significantly outperformed conventional top-down mass spectrometry methods. For a glycosylated 50.8 kDa antibody heavy chain, sequence coverage was greatly increased, from 27.5% to 83.3%, in 10 minutes of acquisition using a single fragmentation mode. Coverage of the middle 350 residues improved from 0% to >95%, demonstrating near-complete coverage of the difficult-to-characterize internal region of a large protein. The fragmentation patterns of DMTm were found to be complementary to conventional top-down, with higher internal coverage for DMTm and higher terminal coverage for conventional. Accordingly, aggregation of the data from the two modes further increased heavy chain sequence coverage to 90.2%. A new software platform, Proteoform Studio, provided optimized ion processing for improved sequence coverage and enabled real-time experimental monitoring as individual ions were accumulated. The platform automatically integrates conventional and DMTm data to provide the most comprehensive sequence coverage possible. Together, these advances enable substantially deeper proteoform sequencing and establish a straightforward, complete top-down DMTm workflow to confidently define proteoforms in biological systems and biotherapeutic development.

bioinformatics↗

bioRxiv2024

Specialized S-type ribosomes of Plasmodium yoelii enhance host-to-vector malaria transmission

Unlike most eukaryotes, Plasmodium species only encode 4-5 ribosomal DNA loci, making them an exceptional model system for genetic studies of ribosome specialization. Plasmodium ribosomes are characterized as A-type and S-type, defined by differing temporal expression patterns and sequence variation. The two S-type rRNAs (S1 & S2) are most abundant in developing mosquito stages, yet maintain low levels of abundance in blood stages. Two previous, conflicting studies found that one or either S-type rRNA was essential to mosquito-stage development, but these experiments were hampered by technical constraints. Therefore, we used the DiCre recombinase to generate transgenic parasites with clean deletions of the S1, S2, or both S-type rDNAs (S-type null) to characterize their roles and interplay in both mosquito- and blood-stage parasites. Contrary to previous conclusions, the presence of either or both S-type rRNAs was not required for sporozoite development, yet promoted host-to-vector transmission, oocyst maturation, and productive sporogony. Unexpectedly, we found that S-type ribosomes have different, opposing impacts on blood stage development despite being at low abundances. Deletion of S1 rDNA nearly ablated the first wave of parasitemia without impacting transmissible male gametocyte counts. Reciprocally, the deletion of S2 rDNA reduced counts of transmissible male gametocytes without impacting parasitemia, which was phenocopied by the S-type null line. Because the LSU portion of S1 rDNA could not be deleted, we assessed the S-type SSU rDNAs for potentially dominant roles. Introduction of extra plasmid-based copies of either S1 or S2 SSU rDNA resulted in either increased parasitemia or male gametogenesis, respectively. Yet, both decreased transmission and impacted early mosquito-stage development. These results indicate that driving functions of Plasmodiums S-type ribosomes occur during blood stages, with S-type SSUs contributing distinct, separable functions. To the best of our knowledge, this is the first example of specialized ribosomes driving cellular outcomes at low abundances. Author SummaryMalaria parasites (Plasmodium spp.) have specialized ribosomes with distinct temporal expression patterns and ribosomal RNA (rRNA) sequence variation. While previous work has demonstrated the importance of the S1 or S2 types of ribosomes to mosquito-stage development, technical limitations led to conflicting conclusions and prevented the investigation of whether the S-type ribosomes were essential for parasite development. Here, we used a DiCre recombinase system to overcome past limitations and generated clean deletions of both S-type rDNA genes, as well as the first Plasmodium line with both S-type rDNA genes deleted. Surprisingly, we found that Plasmodium yoelii parasites do not require either S-type ribosome for mosquito-stage development, when these rRNAs are most abundant. Instead, we found that S-type ribosomes contribute distinct functions during blood-stage development when these ribosomes make up only a small fraction of the total ribosomes present. In these stages, we found that S1 and S2 ribosomes have different, opposing functions that work cooperatively to promote transmission from the mammalian host to the mosquito vector.

microbiology↗

bioRxiv2024

PfCAP-H is essential for assembly of condensin I complex and karyokinesis during asexual proliferation of Plasmodium falciparum

Condensin I is a pentameric complex that regulates the mitotic chromosome assembly in eukaryotes. The kleisin subunit CAP-H of the condensin I complex acts as a linchpin to maintain the structural integrity and loading of this complex on mitotic chromosomes. This complex is present in all eukaryotes and has recently been identified in Plasmodium spp. However, how this complex is assembled and whether the kleisin subunit is critical for this complex in these parasites is yet to be explored. To examine the role of PfCAP-H during cell division within erythrocytes, we generated an inducible PfCAP-H knockout parasite. We find that PfCAP-H is dynamically expressed during mitosis with the peak expression at the metaphase plate. PfCAP-H interacts with PfCAP-G and is a non-SMC member of the condensin I complex. Notably, the absence of PfCAP-H does not alter the expression of PfCAP-G but affects its localization at the mitotic chromosomes. While mitotic spindle assembly is intact in PfCAP-H deficient parasites, duplicated centrosomes remain clustered over the mass of unsegmented nuclei with failed karyokinesis. This failure leads to the formation of an abnormal nuclear mass, while cytokinesis occurs normally. Altogether, our data suggest that PfCAP-H plays a crucial role in maintaining the structural integrity of the condensin I complex on the mitotic chromosomes and is essential for the asexual development of malarial parasites. ImportanceMitosis is a fundamental process for Plasmodium parasites, which plays a vital role in their survival within two distinct hosts - human and Anopheles mosquitoes. Despite its great significance, our comprehension of mitosis and its regulation remains limited. In eukaryotes, mitosis is regulated by one of the pivotal complexes known as condensin complexes. The condensin complexes are responsible for chromosome condensation, ensuring the faithful distribution of genetic material to daughter cells. While condensin complexes have recently been identified in Plasmodium spp, our understanding of how this complex is assembled and their precise functions during the blood stage development of Plasmodium falciparum remains largely unexplored. In this study, we investigate the role of a central protein, PfCAP-H, during the blood stage development of P. falciparum. Our findings reveal that PfCAP-H is essential and plays a pivotal role in upholding the structure of condensin I and facilitating karyokinesis.

cell biology↗

bioRxiv2024

Global Release of Translational Repression Across Plasmodiums Host-to-Vector Transmission Event

Malaria parasites must be able to respond quickly to changes in their environment, including during their transmission between mammalian hosts and mosquito vectors. Therefore, before transmission, female gametocytes proactively produce and translationally repress mRNAs that encode essential proteins that the zygote requires to establish a new infection. This essential regulatory control requires the orthologues of DDX6 (DOZI), LSM14a (CITH), and ALBA proteins to form a translationally repressive complex in female gametocytes that associates with many of the affected mRNAs. However, while the release of translational repression of individual mRNAs has been documented, the details of the global release of translational repression have not. Moreover, the changes in spatial arrangement and composition of the DOZI/CITH/ALBA complex that contribute to translational control are also not known. Therefore, we have conducted the first quantitative, comparative transcriptomics and DIA-MS proteomics of Plasmodium parasites across the host-to-vector transmission event to document the global release of translational repression. Using female gametocytes and zygotes of P. yoelii, we found that nearly 200 transcripts are released for translation soon after fertilization, including those with essential functions for the zygote. However, we also observed that some transcripts remain repressed beyond this point. In addition, we have used TurboID-based proximity proteomics to interrogate the spatial and compositional changes in the DOZI/CITH/ALBA complex across this transmission event. Consistent with recent models of translational control, proteins that associate with either the 5 or 3 end of mRNAs are in close proximity to one another during translational repression in female gametocytes and then dissociate upon release of repression in zygotes. This observation is cross-validated for several protein colocalizations in female gametocytes via ultrastructure expansion microscopy and structured illumination microscopy. Moreover, DOZI exchanges its interaction from NOT1-G in female gametocytes to the canonical NOT1 in zygotes, providing a model for a trigger for the release of mRNAs from DOZI. Finally, unenriched phosphoproteomics revealed the modification of key translational control proteins in the zygote. Together, these data provide a model for the essential translational control mechanisms used by malaria parasites to promote their efficient transmission from their mammalian host to their mosquito vector.

microbiology↗

bioRxiv2021

Direct Imaging and Identification of Proteoforms up to 70 kDa from Human Tissue

Imaging of proteoforms in human tissues is hindered by low molecular specificity and limited proteome coverage. Here, we introduce proteoform imaging mass spectrometry (PiMS), which increases the size limit for proteoform detection and identification by 4-fold compared to reported methods, and reveals tissue localization of proteoforms at <80 m spatial resolution. PiMS advances proteoform imaging by combining ambient nanospray desorption electrospray ionization (nano-DESI) with ion detection using individual ion mass spectrometry (I2MS). We demonstrate the first proteoform imaging of human kidney, identifying 169 of 400 proteoforms <70 kDa using top-down mass spectrometry and database lookup from the human proteoform atlas, including dozens of key enzymes in primary metabolism. PiMS images reveal distinct spatial localizations of proteoforms to both anatomical structures and cellular neighborhoods in the vasculature, medulla, and cortex regions of the human kidney. The benefits of PiMS are poised to increase proteome coverage for label-free protein imaging of tissues. TeaserNano-DESI combined with individual ion mass spectrometry generates images of proteoforms up to 70 kDa.

biochemistry↗

bioRxiv2020

Standard Procedures for Native CZE-MS of Proteins and Protein Complexes up to 800 kDa

Native mass spectrometry (nMS) is a rapidly growing method for the characterization of large proteins and protein complexes, preserving "native" non-covalent inter- and intramolecular interactions. Direct infusion of purified analytes into a mass spectrometer represents the standard approach for conducting nMS experiments. Alternatively, CZE can be performed under native conditions, providing high separation performance while consuming trace amounts of sample material. Here, we provide standard operating procedures for acquiring high quality data using CZE in native mode coupled online to various Orbitrap mass spectrometers via a commercial sheathless interface, covering a wide range of analytes from 30 - 800 kDa. Using a standard protein mix, the influence of various CZE method parameters were evaluated, such as BGE/conductive liquid composition and separation voltage. Additionally, a universal approach for the optimization of fragmentation settings in the context of protein subunit and metalloenzyme characterization is discussed in detail for model analytes. A short section is dedicated to troubleshooting of the nCZE-MS setup. This study is aimed to help normalize nCZE-MS practices to enhance the CE community and provide a resource for production of reproducible and high-quality data.

biochemistry↗

bioRxiv2019

Direct Measurement of Light and Heavy Antibody Chains Using Differential Ion Mobility Spectrometry and Middle-Down Mass Spectrometry

The analysis of monoclonal antibodies (mAbs) by a middle-down approach is a growing field that attracts the attention of many researchers and biopharma companies. Usually, liquid fractionation techniques are used to separate mAbs polypeptides chains before mass spectrometry (MS) analysis. Gas-phase fractionation techniques such as high-field asymmetric waveform ion mobility spectrometry (FAIMS) can replace liquid-based separations and reduce both analysis time and cost. Here, we present a rapid FAIMS tandem MS method capable of characterizing the polypeptide sequence of mAbs light (Lc) and heavy (Hc) chains in an unprecedented, easy, and fast fashion. This new method uses commercially available instruments and takes [~] 24 minutes --40-60% faster than regular LC-MS/MS analysis -- to acquire fragmentation data using different dissociation methods.

biochemistry↗