Concise review on the combined use of immunocapture, mass spectrometry and liquid chromatography for clinical applications.

Immunocapture is now a well-established method for sample preparation prior to quantitation of peptides and proteins in complex matrices. This short review will give an overview of some clinical applications of immunocapture methods, as well as protocols with and without enzymatic digestion in a clinical context. The advantages and limitations of both approaches are discussed in detail. Challenges related to the choice of mass spectrometer are also discussed. Top-down, middle-down, and bottom-up approaches are discussed. Even though immunocapture has its limitations, its main advantage is that it provides an additional dimension of separation and/or isolation when working with peptides and proteins. Overall, this short review demonstrates the potential of such techniques in the field of proteomics-based clinical medicine and paves the way for better personalized medicine.

Daytime rest: Association with 24-h rest-activity cycles, circadian timing and cognition in older adults.

Growing epidemiological evidence points toward an association between fragmented 24-h rest-activity cycles and cognition in the aged. Alterations in the circadian timing system might at least partially account for these observations. Here, we tested whether daytime rest (DTR) is associated with changes in concomitant 24-h rest probability profiles, circadian timing and neurobehavioural outcomes in healthy older adults. Sixty-three individuals (59-82 years) underwent field actigraphy monitoring, in-lab dim light melatonin onset assessment and an extensive cognitive test battery. Actimetry recordings were used to measure DTR frequency, duration and timing and to extract 24-h rest probability profiles. As expected, increasing DTR frequency was associated not only with higher rest probabilities during the day, but also with lower rest probabilities during the night, suggesting more fragmented night-time rest. Higher DTR frequency was also associated with lower episodic memory performance. Moreover, later DTR timing went along with an advanced circadian phase as well as with an altered phase angle of entrainment between the rest-activity cycle and circadian phase. Our results suggest that different DTR characteristics, as reflective indices of wake fragmentation, are not only underlined by functional consequences on cognition, but also by circadian alteration in the aged.

Development and validation of an LC-MS/MS method for the simultaneous quantitation of angiotensin (1-7), (1-8), (1-9) and (1-10) in human plasma.

Cardiovascular diseases have cast a significant negative impact on the lives of millions worldwide. Over the years, extensive efforts have been dedicated to enhancing diagnostic and prognostic tools for these diseases. A growing body of evidence indicates that the angiotensin convertase enzyme (ACE) and the angiotensin convertase enzyme 2 (ACE2), and angiotensin peptide levels could hold a pivotal role in assisting clinicians with the management of cardiovascular conditions, notably hypertension and heart failure. However, despite the considerable body of knowledge in this domain, a void remains in the field of analytical methodologies for these molecules. In this study, we present a fully validated LC-MS/MS method for the precise quantitation of plasma angiotensin (1-7), (1-8), (1-9), and (1-10), following the guidelines set by the Clinical and Laboratory Standards Institute (CLSI). Our method not only enables the accurate quantification of angiotensin peptides but also provides a means to assess ACE and ACE2 activity. Remarkably, our method achieved a Lower Limit of Measurement Interval (LLMI) as low as 5 pg/mL. This has enabled the detection of angiotensin (1-7), (1-8), (1-9) and (1-10) and the accurate quantitation of angiotensin (1-7), (1-8) and (1-10) in all analyzed groups, including healthy controls, patients with high blood pressure, and patients with chronic kidney disease. To our knowledge, our method represents the most sensitive approach allowing for simultaneous quantitation of these four angiotensin peptides. A distinct advantage of our method, when compared to immunoassays, is its high sensitivity combined with comprehensive chromatographic separation of all currently known angiotensin peptides. This combination translates to an exceptional level of selectivity, underscoring the value and potential of our methodology in advancing cardiovascular disease research.

Development and Validation of an Ultrasensitive LC-MS/MS Method for the Quantification of Melatonin in Human Saliva.

A growing body of literature describes the potential effects of circadian disruption on human health. Indeed, psychiatric diseases, metabolic syndrome, and cancers may be linked to disturbance of the circadian rhythm. Currently, the best practice to assess circadian rhythm is the measurement of melatonin levels. Our goal was thus to develop and validate a highly sensitive LC-MS/MS method to follow salivary melatonin levels throughout the day and night. Our method reached a lower limit of the measuring interval (LLMI) of 0.8 pg/mL. To our knowledge, it is the most sensitive method allowing quantitation of melatonin in saliva. Saliva, obtained from passive drooling or salivette, was extracted by an efficient and quick liquid-liquid extraction with no further cleanup needed. The method was validated according to the European Medicines Agency (EMA) guidelines and provided excellent results regarding accuracy, precision, linearity, selectivity, and specificity. Comparison between radioimmunoassay and our method was performed and showed differences at low levels, most likely due to cross-reactivity with other indols. To assess daytime melatonin levels in humans, salivary melatonin levels of ten volunteers were monitored throughout the day and showed lower daytime levels than reported in previous studies.

Innovative workflow for the identification of cathepsin K cleavage sites in type I collagen.

Since the late 1990s, cathepsin K cleavage sites in type I collagen have been extensively studied due to its ability to release bone resorption biomarkers such as CTX and NTX. However, gel-based methods and N-sequencing used in these studies lack sensitivity, especially for small to medium peptides. In this work, we propose a degradomics mass spectrometry-based workflow that combines protein digestion, Nano-LC-UDMSE, and several software tools to identify cathepsin K cleavage sites. This workflow not only identified previously known cleavage sites, but also discovered new ones. Multiple cleavage hotspots were found and described in type I α1 and type I α2 collagen, many of which coincided with pyridinoline crosslinks, known to stabilize the triple helix. Our results allowed us to establish a chronology of digestion and conclude that cathepsin K preferentially cleaves the extremities of type I collagen before the helical part. We also found that cathepsin K preferentially cleaves amino acid residues with long and hydrophobic lateral chains at the beginning of digestion, whereas no preferred amino acid residues were identified later in the digestion. In conclusion, our workflow successfully identified new cleavage sites and can be easily applied to other proteins or proteases.