The One Hour Human Proteome.

We describe deep analysis of the human proteome in less than 1 h. We achieve this expedited proteome characterization by leveraging state-of-the-art sample preparation, chromatographic separations, and data analysis tools, and by using the new Orbitrap Astral mass spectrometer equipped with a quadrupole mass filter, a high-field Orbitrap mass analyzer, and an asymmetric track lossless (Astral) mass analyzer. The system offers high tandem mass spectrometry acquisition speed of 200 Hz and detects hundreds of peptide sequences per second within data-independent acquisition or data-dependent acquisition modes of operation. The fast-switching capabilities of the new quadrupole complement the sensitivity and fast ion scanning of the Astral analyzer to enable narrow-bin data-independent analysis methods. Over a 30-min active chromatographic method consuming a total analysis time of 56 min, the Q-Orbitrap-Astral hybrid MS collects an average of 4319 MS1 scans and 438,062 tandem mass spectrometry scans per run, producing 235,916 peptide sequences (1% false discovery rate). On average, each 30-min analysis achieved detection of 10,411 protein groups (1% false discovery rate). We conclude, with these results and alongside other recent reports, that the 1-h human proteome is within reach.

Crowd control of ions in the Astral analyzer.

Space charge effects are the Achilles' heel of all high-resolution ion optical devices. In time-of-flight mass analyzers, these may manifest as reduction of resolving power, mass measurement shift, peak coalescence, and/or transmission losses, while highly sensitive modern ion sources and injection devices ensure that such limits are easily exceeded. Space charge effects have been investigated, by experiment and simulation study, for the astral multi-reflection analyzer, incorporating ion focusing via a pair of converging ion mirrors, and fed by a pulsed extraction ion trap. Major factors were identified as the resonant effect between ~103 ions of similar m/z in-flight and the expansion of trapped packets of ~104-5 ions prior to extraction. Optimum operation and compensated ion mirror calibration strategies were then generated and described based on these findings.

Ultra-fast label-free quantification and comprehensive proteome coverage with narrow-window data-independent acquisition.

Mass spectrometry (MS)-based proteomics aims to characterize comprehensive proteomes in a fast and reproducible manner. Here we present the narrow-window data-independent acquisition (nDIA) strategy consisting of high-resolution MS1 scans with parallel tandem MS (MS/MS) scans of ~200 Hz using 2-Th isolation windows, dissolving the differences between data-dependent and -independent methods. This is achieved by pairing a quadrupole Orbitrap mass spectrometer with the asymmetric track lossless (Astral) analyzer which provides >200-Hz MS/MS scanning speed, high resolving power and sensitivity, and low-ppm mass accuracy. The nDIA strategy enables profiling of >100 full yeast proteomes per day, or 48 human proteomes per day at the depth of ~10,000 human protein groups in half-an-hour or ~7,000 proteins in 5 min, representing 3× higher coverage compared with current state-of-the-art MS. Multi-shot acquisition of offline fractionated samples provides comprehensive coverage of human proteomes in ~3 h. High quantitative precision and accuracy are demonstrated in a three-species proteome mixture, quantifying 14,000+ protein groups in a single half-an-hour run.

A Conjoined Rectilinear Collision Cell and Pulsed Extraction Ion Trap with Auxiliary DC Electrodes.

Ion traps are routinely directly coupled to mass analyzers, where they serve to suitably cool and shape an ion population prior to pulsed extraction into the analyzer proper. Such devices benefit from high duty cycle and transmission but suffer slow ion processing times caused by a compromise in the buffer gas pressure range that suitably dampens the ion kinetic energy without causing excessive scatter during extraction or within the analyzer. A rectilinear RF quadrupole ion trap has been characterized, conjoining a pressurized collision region with a pumped extraction region, and an unbroken RF interface for seamless ion transfer between them. Auxiliary electrodes mounted between the RF electrodes provide DC voltage gradients that serve to both guide ions through the device and position them at the extraction slot. The influence of the auxiliary DC upon the trapping RF field was measured, and suitable parameters were defined. A mode of operation was developed that allowed parallel processing of ions in both regions, enabling a repetition rate of 200 Hz when the device was coupled to a high-resolution accurate-mass analyzer.

Fast and Deep Phosphoproteome Analysis with the Orbitrap Astral Mass Spectrometer.

Owing to its roles in cellular signal transduction, protein phosphorylation plays critical roles in myriad cell processes. That said, detecting and quantifying protein phosphorylation has remained a challenge. We describe the use of a novel mass spectrometer (Orbitrap Astral) coupled with data-independent acquisition (DIA) to achieve rapid and deep analysis of human and mouse phosphoproteomes. With this method we map approximately 30,000 unique human phosphorylation sites within a half-hour of data collection. We applied this approach to generate a phosphoproteome multi-tissue atlas of the mouse. Altogether, we detected 81,120 unique phosphorylation sites within 12 hours of measurement. With this unique dataset, we examine the sequence and structural context of protein phosphorylation. Finally, we highlight the discovery potential of this resource with multiple examples of novel phosphorylation events relevant to mitochondrial and brain biology.

Parallelized Acquisition of Orbitrap and Astral Analyzers Enables High-Throughput Quantitative Analysis.

The growing trend toward high-throughput proteomics demands rapid liquid chromatography-mass spectrometry (LC-MS) cycles that limit the available time to gather the large numbers of MS/MS fragmentation spectra required for identification. Orbitrap analyzers scale performance with acquisition time and necessarily sacrifice sensitivity and resolving power to deliver higher acquisition rates. We developed a new mass spectrometer that combines a mass-resolving quadrupole, the Orbitrap, and the novel Asymmetric Track Lossless (Astral) analyzer. The new hybrid instrument enables faster acquisition of high-resolution accurate mass (HRAM) MS/MS spectra compared with state-of-the-art mass spectrometers. Accordingly, new proteomics methods were developed that leverage the strengths of each HRAM analyzer, whereby the Orbitrap analyzer performs full scans with a high dynamic range and resolution, synchronized with the Astral analyzer's acquisition of fast and sensitive HRAM MS/MS scans. Substantial improvements are demonstrated over previous methods using current state-of-the-art mass spectrometers.

Evaluating the Performance of the Astral Mass Analyzer for Quantitative Proteomics Using Data-Independent Acquisition.

We evaluate the quantitative performance of the newly released Asymmetric Track Lossless (Astral) analyzer. Using data-independent acquisition, the Thermo Scientific Orbitrap Astral mass spectrometer quantifies 5 times more peptides per unit time than state-of-the-art Thermo Scientific Orbitrap mass spectrometers, which have long been the gold standard for high-resolution quantitative proteomics. Our results demonstrate that the Orbitrap Astral mass spectrometer can produce high-quality quantitative measurements across a wide dynamic range. We also use a newly developed extracellular vesicle enrichment protocol to reach new depths of coverage in the plasma proteome, quantifying over 5000 plasma proteins in a 60 min gradient with the Orbitrap Astral mass spectrometer.

Evaluating the Performance of the Astral Mass Analyzer for Quantitative Proteomics Using Data Independent Acquisition.

We evaluate the quantitative performance of the newly released Asymmetric Track Lossless (Astral) analyzer. Using data independent acquisition, the Thermo Scientific™ Orbitrap™ Astral™ mass spectrometer quantifies 5 times more peptides per unit time than state-of-the-art Thermo Scientific™ Orbitrap™ mass spectrometers, which have long been the gold standard for high resolution quantitative proteomics. Our results demonstrate that the Orbitrap Astral mass spectrometer can produce high quality quantitative measurements across a wide dynamic range. We also use a newly developed extra-cellular vesicle enrichment protocol to reach new depths of coverage in the plasma proteome, quantifying over 5,000 plasma proteins in a 60-minute gradient with the Orbitrap Astral mass spectrometer.

The association of Ned Kelly tattoos with suicide and homicide in a forensic context-a confirmatory prospective study.

Ned Kelly, an iconic figure in contemporary Australian mythology, was a bushranger (outlaw) who was executed in 1880 for the murder of a serving police officer, Constable Thomas Lonigan. Kelly is often commemorated by tattoos which depict his armour and helmet or his alleged last words of "Such is life". A study was undertaken from January 1, 2011, to December 31, 2020, at Forensic Science SA, Adelaide, South Australia, of all cases with such tattoos. De-identified case details included the year of death, age, sex and cause and manner of death. There were 38 cases consisting of 10 natural deaths (26.3%) and 28 unnatural (73.7%). The latter included 15 cases of suicide (39.5%), 9 accidents (23.7%) and 4 homicides (10.5%). Of the 19 suicides and homicides, there were 19 males and no females (age range 24-57 years; average 44 years). The number of suicides in the general South Australian forensic autopsy population in 2020 was 216/1492 (14.5%) which was significantly lower than in the study population in which 39.5% of cases were suicides (2.7 times higher; p < 0.001). A similar trend occurred for homicides which accounted for 17/1492 in the general forensic autopsy population (1.1%), significantly lower than in the study population which had 10.5% homicides (approximately 9.5 times higher; p < 0.001). Thus, in the select population referred for medicolegal autopsy, there appears no doubt that Ned Kelly tattoos are associated with suicides and homicides. While this is not a population-based study, it may provide useful information for forensic practitioners dealing with such cases.

Scurvy-Characteristic Features and Forensic Issues.

Scurvy is a multisystem condition that arises from vitamin C deficiency. As humans cannot synthesize vitamin C, serum and tissue levels depend on bioavailability, utilization, and renal excretion. Deficiencies result in defective collagen formation with swelling of gums, leg ulceration, and bleeding manifestations. Death most often results from infection and hemorrhage. In a forensic context, scurvy may mimic inflicted injuries and may be responsible for sudden death by mechanisms that remain unclear. Cardiac failure and rhythm disturbances with chest pain, hypotension, cardiac tamponade, and dyspnea are associated with vitamin C deficiency. In addition, syncope and seizures may occur. Although far less common than in previous centuries, scurvy is still present in high-risk populations that include alcoholics, isolated elderly individuals, food faddists, institutionalized patients, those with mental illness, and those who have had bariatric surgery or with underlying gastrointestinal conditions. Scurvy should therefore be a diagnosis to consider in medicolegal cases of apparent trauma and sudden death.

Judicial Murder-Suicides in Van Diemen's Land.

On the morning of December 17, 1827, nine convicts were executed by public hanging in Hobart Town, the capital of the British colony of Van Diemen's Land (now the Australian state of Tasmania). Two months previously they had drowned senior Constable George Rex on Small Island, which was part of the penal settlement at Macquarie Harbor, in front of five bound and gagged witnesses. They offered no defence at their trial. Examination of the Tasmanian colonial convict records shows that "suicide by lottery" involved convicts choosing two men, one to die and the other to kill him. The witnesses would earn a respite when taken away for the trial, and the murderer would be executed. "Death by gallows" could be considered a nineteenth-century version of an orchestrated suicide reminiscent of more modern "death by cop." This category of "judicial" murder-suicide expands the range of contemporary classifications of dyadic deaths.

Cannibalism amongst penitentiary escapees from Sarah Island in nineteenth century Van Diemen's Land.

Alexander Pearce was an Irish convict incarcerated on Sarah Island on the west coast of Van Diemen's Land (modern day Tasmania, Australia) in 1822, following his transportation to the colony from the United Kingdom for seven years in 1819. On two occasions he escaped from the island, in September 1822 and again in November 1823, and was only able to survive the harsh conditions by killing and consuming his fellow escapees. Given that Pearce utilized the only sustenance that was at hand (i.e. his five companions), and that there was a temporal separation between the two episodes, this may represent a separate category of anthropophagy, that of serial opportunistic cannibalism.

Activation of Methane by the Pyridine Radical Cation and its Substituted Forms in the Gas Phase.

We present an experimental study of methane activation by pyridine cation and its substituents in the gas phase. Mass spectrometric experiments in an ion trap demonstrate that pyridine cation and some of its substituent cations are able to react with methane. The deuterated methane experiment has confirmed that the hydrogen atom in the ionic product of reaction does come from methane. The collected information about kinetic isotope effects has been used to distinguish the nature of the bond activation as a hydrogen abstraction. Furthermore, experimental results demonstrated that the substituent groups on the pyridine ring can crucially influence their reactivity in methane bond activation processes. Density functional calculation (DFT) was employed to study the electronic structures of the complex and reaction mechanism of CH4+C5H5N(+). The calculations confirmed the hypothesis from the experimental observation, namely, the reaction is rapid with no energy barrier.

Ultraviolet photofragmentation spectroscopy of alkaline earth dication complexes with pyridine and 4-picoline (4-methyl pyridine).

A detailed experimental and theoretical study has been undertaken of the UV photofragmentation spectroscopy of the alkaline earth metal dications Mg(2+), Ca(2+), and Sr(2+) complexed with pyridine and 4-methyl pyridine (4-picoline). The ion complexes have been prepared using the pick-up technique and held in an ion trap where their internal temperature has been reduced to <150 K. Exposure of the trapped ions to tunable UV laser radiation leads to the appearance of photofragments with intensities that show significant variation as a function of wavelength. For all three metal dications, the resultant spectra show evidence of resolved features. Time-dependent density functional theory (TDDFT) has been used to identify possible electronic transitions that might be present in the [M(pyridine)(4)](2+) complexes (M = Mg, Ca, and Sr) within the wavelength range studied. These calculations show that the spectra are dominated by strong π* ← π and weaker π* ← n transitions localized on the pyridine ligands. The calculations correctly identify those regions of the experimental spectra where UV transitions begin to occur in the complexes and also the wavelengths at which absorption maxima are reached; however, more subtle features of the spectra are difficult to assign with confidence.

State-resolved UV photofragmentation spectrum of the metal dication complex [Zn(pyridine)(4)](2+).

A combined theoretical and experimental study of electronic transitions in the complex [Zn(pyridine)(4)](2+) provides the first example of a state-resolved electronic spectrum to be recorded for a dication complex in the gas phase.