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The Hunt Lab Guide to De Novo Peptide Sequence Analysis by Tandem Mass Spectrometry.

Donald Hunt has made seminal contributions to the fields of proteomics, immunology, epigenetics, and glycobiology. The foundation of every important work to come out of the Hunt Laboratory is de novo peptide sequencing. For decades, he taught hundreds of students, postdocs, engineers, and scientists to directly interpret mass spectral data. To honor his legacy and ensure that the art of de novo sequencing is not lost, we have adapted his teaching materials into "The Hunt Lab Guide to De Novo Peptide Sequence Analysis by Tandem Mass Spectrometry". In addition to the de novo sequencing tutorials, we present two freely available software tools that facilitate manual interpretation of mass spectra and validation of search results. The first, "Hunt Lab Peptide Fragment Calculator", calculates precursor and fragment mass-to-charge ratios for any peptide. The second program, "Predator Protein Fragment Calculator", was inspired in part by the fragment calculator developed in the Hunt Lab. Its capabilities are enhanced to facilitate interpretation of mass spectral data derived from intact proteins. We hope that the combination of these educational tools will continue to benefit students and researchers by empowering them to interpret data on their own.

Tandem Mass Spectrometry

Mass Spectrometry-Based Proteomics for the Masses: Peptide and Protein Identification in the Hunt Laboratory During the 2000's.

There has been a rapid increase in the number of individuals utilizing mass spectrometry-based proteomics to study complex biological systems and questions since the start of the 2000's. Building off the advancements in ionization and liquid chromatography scientists continued to push towards technology that would enable in-depth analysis of biological specimen. Donald F Hunt and the Hunt laboratory were major contributors to this effort with their work on improving upon existing Fourier Transform MS, development of electron transfer dissociation, and continued work on ion-ion reactions to improve intact protein analysis. Collaboration with other instrumentation laboratories and instrument companies led to the sharing of technology and eventual commercialization providing greater access. Additionally, the Hunt laboratory spread the gospel of MS-based proteomics through collaborations that lasted decades with other scientists who were experts in immunology, cellular signaling, epigenetics, and other fascinating fields. This article attempts to highlight the many contributions of Don and the Hunt laboratory to peptide and protein identification since the year 2000.

Humans

On the Hunt for the Histone Code.

Our genome is not made of naked DNA but a fiber (chromatin) composed of DNA and proteins packaged into our chromosomes. The basic building block of chromatin is the nucleosome, which has two copies of each of the proteins called histones (H2A, H2B, H3, and H4) wrapped by 146 base pairs of DNA. Regions of our genetic material are found between the more open (euchromatin) and more compact (heterochromatin) regions of the genome that can be variably accessible to the underlying genes. Furthermore, post-translational modifications (PTMs) on histones, such as on H3, are critical for regulating chromatin accessibility and gene expression. While site-specific antibodies were the tool of choice for histone PTM analysis in the early days (pre-2000s), enter Don Hunt changing the histone PTM field forever. Don's clever thinking brought new innovative mass spectrometry-based approaches to the epigenetics field. His lab's effort led to the discovery of many new histone modifications and methods to facilitate the detection and quantification of histone PTMs, which are still considered state of the art in the proteomics field today. Due to Don's pioneering work in this area, many labs have been able to jump into the epigenetics field and "Hunt" down their own histone targets. A walkthrough of those early histone years in the Hunt Lab is described by three of us who were fortunate enough to be at the right place, at the right time.

Animals

A Donald F. Hunt Story (John's Version).

A personal narrative of my time in the Hunt laboratory and beyond is provided. The impact of the Hunt laboratory on the analysis of peptides and proteins by tandem mass spectrometry is described in the context of the time.

History, 20th Century

Brain damage in National Hunt jockeys.

Five National Hunt jockeys have been found to have post-traumatic encephalopathy- three with epilepsy and two with significant intellectual and psychological deterioration. Closer supervision is needed.

Adult

Orbital venography in painful ophthalmoplegia (Tolosa-Hunt syndrome).

Twenty-six patients with painful ophthalmoplegia were classified as having the Tolosa-Hunt syndrome on the basis of their clinical findings and response to steroid therapy. All patients underwent orbital venography, a review of which forms the contents of this investigation. Orbital venography, a review of which forms the contents of this investigation. Orbital venograms were normal in 16 patients and abnormal in 10. The major abnormalities were (1) obstruction of the superior ophthalmic vein in its third segment without displacement; (2) collateral venous flow through small venous channels or collateral veins; and (3) poor opacification of the ipsilateral cavernous sinus.

Adolescent

Reconstruction after total gastrectomy: construction of a Hunt-Lawrence pouch using Auto Suture staples.

A simple gastric reservoir using autosuture staples can be constructed rapidly after total gastrectomy. The importance of both a reservoir capacity and delayed evacuation was supported by the clinical course of most of the patients. All 18 patients had improved nutrition. Patients resected for cure tended to have greater weight gain and less abdominal discomfort. Occasionally patients who underwent only palliative resection were long-term survivors with a clinical course similar to that of patients resected for cure. On the basis of these results, it is unfair not to consider this type of reconstruction in patients who have undergone a total gastrectomy because it adds little to the operating time, yet it provides better palliation in terms of nutrition and abdominal comfort on both a short- and long-term basis.

Gastrectomy

The hunt for renal hypertension.

The proportion of cases of hypertension with a renal cause which can be corrected by surgery is very small. Radiological diagnosis of such a cause is expensive and time-consuming; hence the rewards of intensive investigation are small compared with the cost. Measurement of plasma-renin offers a good method of selecting those patients who require further investigation, if the cut-off point is carefully considered in the light of the acceptable incidence of false-positive and false-negative results.

Cost-Benefit Analysis

Bounty hunting.

Explore the source record for details and available documents.

Audiovisual Aids

Patterns of Genetic Diversity Within Three California Quail Species Are Best Explained by Climate and Landscape Changes.

Many North American game animals experienced severe population declines during the 19th century due to market hunting. However, estimates of the timing and magnitude of these declines often rely on anecdotal evidence, which makes it difficult to understand the lasting impacts of hunting pressures versus climate or landscape changes on the genetic diversity of contemporary populations. Historical reports suggest the California quail (Callipepla californica) suffered more significant hunting pressure in the late 19th century relative to either Gambel's (Callipepla gambelii) or mountain quail (Oreortyx pictus). Genomic data can help illuminate the extent to which historical exploitation moulded the genetic health of modern quail populations. We compared whole genome sequences from these three quail species to evaluate whether reported differences in hunting pressure affected contemporary patterns of genetic diversity. Contrary to our expectations, California quail did not exhibit any evidence for population declines until the late 20th century, long after the era of market hunting ended. California quail also exhibited the highest levels of genetic diversity across most analyses with evidence for population expansion over the past 500,000 years. In contrast, the mountain quail exhibited a long-term population decline beginning in the middle of the last ice age 30-40 thousand years ago. The Gambel's quail appears to have suffered a more recent bottleneck in association with a major drought that impacted the desert southwest during the mid-20th century. Gambel's quail also exhibited increased realised genetic load for mild and moderately deleterious genetic variants. Together, our results demonstrate that market hunting had little lasting impact on the genetic diversity of these quail species, whereas landscape and climate changes have led to fluctuations in effective population size (Ne) and the buildup of genetic load.

Animals

Innovations Toward Immunopeptidomics.

Over the past 30 years, immunopeptidomics has grown alongside improvements in mass spectrometry technology, genomics, transcriptomics, T cell receptor sequencing, and immunological assays to identify and characterize the targets of activated T cells. Together, multiple research groups with expertise in immunology, biochemistry, chemistry, and peptide mass spectrometry have come together to enable the isolation and sequence identification of endogenous major histocompatibility complex (MHC)-bound peptides. The idea to apply highly sensitive mass spectrometry techniques to study the landscape of peptide antigens presented by cell surface MHCs was innovative and continues to be successfully used and improved upon to deepen our understanding of how peptide antigens are processed and presented to T cells. Multiple research groups were involved in this bringing immunopeptidomics to the forefront of translational research, and we will highlight the contributions of one of the earliest developers, Professor Donald F. Hunt, and his research group at the University of Virginia. The Hunt laboratory applied cutting edge mass spectroscopy-based immunopeptidomics to study cancer, autoimmunity, transplant rejection, and infectious diseases. Across these diverse research areas, the Hunt laboratory and collaborators would characterize previously unknown MHC peptide-binding motifs and identify immunologically active antigens using ultra sensitive mass spectrometry techniques. Amazingly, many of the MHC-bound peptide antigens discovered in collaborations with the Hunt laboratory were sequenced by mass spectrometry before the completion of the human genome using manual de novo sequencing. In this perspective article, we will chronicle the work of the Hunt laboratory and their many collaborators that would be a major part of the foundation for mass spectrometry-based immunopeptidomics and its application to immunology research.

Animals

First case of Brucella suis biovar 1 infection in a dog in Switzerland.

As one of the most common zoonoses globally, brucellosis threatens not only animals but also humans with Brucella (B.) melitensis, B. abortus and B. suis biovar 1&3, being more virulent for humans than B. canis, B. ovis and B. suis biovar 2. Canine brucellosis is caused mainly by B. canis, however, B. suis infections in dogs have been reported sporadically in Europe and more frequently in Australia. B. suis infection in dogs is mainly associated with hunting or wild-animal exposure and rarely with consumption of commercial raw meat products. In Switzerland, B. canis is sporadically diagnosed in dogs and B. suis biovar 2 is present in wild boar and brown hare. We report a case of brucellosis in a dog from Switzerland, neither having a history of hunting nor travel-associated risk of exposure. The intact male dog showed clinical signs (fever and epididymitis) consistent with brucellosis, which was confirmed by culture and molecular methods in urine and by serological methods. Culture and subsequent whole genome sequencing revealed the isolate as B. suis biovar 1, ST-14. The most closely related strain was shown to be a strain isolated in 2021 from a dog in Germany. Based on the lack of any previous report, infection by B. suis in dogs has not previously been diagnosed in Switzerland. This case highlights the need for vigilance regarding this less expected pathogen of high zoonotic importance in dogs.

Animals