•N-terminal acetylation, pyroglutamate formation, N-degrons and proteolysis are reviewed.•N-terminomics provide comprehensive profiling of modification at protein N-termini in a proteome-wide ...manner.•We outline a number of established methodologies for the enrichment of protein N-termini through positive and negative selection strategies.•Peptidomics-based approach is beneficial for the study of post-translational processing of protein N-termini.
Amino-/N-terminal processing is a crucial post-translational modification affecting almost all proteins. In addition to altering the chemical properties of the N-terminus, these modifications affect protein activation, conversion, and degradation, which subsequently lead to diversified biological functions. The study of N-terminal modifications is of increasing interest; especially since modifications such as proteolytic truncation or pyroglutamate formation have been linked to disease processes. During the past decade, mass spectrometry has played an important role in facilitating the investigation of N-terminal modifications. Continuous progress is being made in the development and application of robust methods for the dedicated analysis of native and modified protein N-termini in a proteome-wide manner. Here we highlight recent progress in our understanding of protein N-terminal biology as well as outlining present enrichment strategies for mass spectrometry-based studies of protein N-termini.
Numerous software tools exist for data-independent acquisition (DIA) analysis of clinical samples, necessitating their comprehensive benchmarking. We present a benchmark dataset comprising real-world ...inter-patient heterogeneity, which we use for in-depth benchmarking of DIA data analysis workflows for clinical settings. Combining spectral libraries, DIA software, sparsity reduction, normalization, and statistical tests results in 1428 distinct data analysis workflows, which we evaluate based on their ability to correctly identify differentially abundant proteins. From our dataset, we derive bootstrap datasets of varying sample sizes and use the whole range of bootstrap datasets to robustly evaluate each workflow. We find that all DIA software suites benefit from using a gas-phase fractionated spectral library, irrespective of the library refinement used. Gas-phase fractionation-based libraries perform best against two out of three reference protein lists. Among all investigated statistical tests non-parametric permutation-based statistical tests consistently perform best.
Effective proteome-wide strategies that distinguish the N-termini of proteins from the N-termini of their protease cleavage products would accelerate identification of the substrates of proteases ...with broad or unknown specificity. Our approach, named terminal amine isotopic labeling of substrates (TAILS), addresses this challenge by using dendritic polyglycerol aldehyde polymers that remove tryptic and C-terminal peptides. We analyze unbound naturally acetylated, cyclized or labeled N-termini from proteins and their protease cleavage products by tandem mass spectrometry, and use peptide isotope quantification to discriminate between the substrates of the protease of interest and the products of background proteolysis. We identify 731 acetylated and 132 cyclized N-termini, and 288 matrix metalloproteinase (MMP)-2 cleavage sites in mouse fibroblast secretomes. We further demonstrate the potential of our strategy to link proteases with defined biological pathways in complex samples by analyzing mouse inflammatory bronchoalveolar fluid and showing that expression of the poorly defined breast cancer protease MMP-11 in MCF-7 human breast cancer cells cleaves both endoplasmin and the immunomodulator and apoptosis inducer galectin-1.
For the simulation of winter hydrological processes a gap in the availability of flow models existed: one either had the choice between (1) physically‐based and fully‐integrated, but computationally ...very intensive, or (2) simplified and compartamentalized, but computationally less expensive, simulators. To bridge this gap, we here present the integration of a computationally efficient representation of winter hydrological processes (snowfall, snow accumulation, snowmelt, pore water freeze–thaw) in a fully‐integrated surface water‐groundwater flow model. This allows the efficient simulation of catchment‐scale hydrological processes in locations significantly influenced by winter processes. Snow accumulation and snowmelt are based on the degree‐day method and pore water freeze–thaw is calculated with a vertical heat conduction approach. This representation of winter hydrological processes is integrated into the fully‐coupled surface water‐groundwater flow model HydroGeoSphere. A benchmark for pore water freeze–thaw as well as two illustrative examples are provided.
Article impact statement: Computationally efficient simulations of catchment‐scale winter hydrological processes in an integrated surface water‐groundwater flow model.
Subjective age, that is the age people feel in relation to their chronological age, can vary on a day-to-day and even momentary basis. Previous long-term and daily-diary studies have shown that ...elevated stress covaries with older subjective age. However, it is an open question whether such links can also be observed at the momentary level within a given day and go beyond self-reports of stress. Moving ahead, we investigated how two indicators of stress (self-reported: perceived stress; physiological: salivary cortisol) are associated with the age people feel on a momentary basis. We examined data from 118 older (Mage = 66.67 years) and 36 very old adults (Mage = 85.92 years) who reported their momentary subjective age and perceived stress and also provided saliva samples up to seven times a day over seven consecutive days. Dynamic structural equation models showed that both higher momentary perceived stress and higher cortisol levels preceding the measurement predicted an older momentary subjective age. In contrast, subjective age at the previous measurement did not predict subsequent stress. These effects were moderated by participant age group and grip strength, albeit not consistently. Our results corroborate and extend earlier findings that both self-reported and physiological stress are important explanatory variables for people's subjective age variation even on relatively short time scales and shed light on differential time-ordered dynamics between stress and subjective age in daily life. Findings also inform theoretical models of subjective age that highlight the importance of contextual, momentary influences on how old people feel and help better understand how biological and psychological processes are intertwined in later life.
Public Significance Statement
This study finds that higher perceived and biological stress affect how older adults feel (i.e., their subjective age) in a given moment: When people are stressed, they feel older. Subjective age is an important predictor of health and well-being in later life. Thus, this study increases our understanding about the factors that might influence how older adults feel and how it translates to outcomes in older adults' daily lives.
Multiple-timescale studies provide new opportunities to examine how developmental processes that evolve at different cadences are intertwined. Developmental theories of emotion regulation suggest ...that the long-term, slowly evolving age-related accumulation of disease burden should shape short-term, faster evolving (daily) affective experiences. To empirically examine this proposition, we combined data from 123 old adults (65-69 years, 47% women) and 32 very old adults (85-88 years, 59% women) who provided 20+ year within-person longitudinal data on physician-rated morbidity and subsequently also completed repeated daily-life assessments of stress and affect six times a day over 7 consecutive days as they were going about their daily-life routines. Results from models that simultaneously articulate growth and intraindividual variability processes (in a dynamic structural equation modeling framework) revealed that individual differences in long-term aging trajectories of the accumulation of disease burden were indeed predictive of differences in three facets of affective dynamics that manifest in everyday life. In particular-over and above mean levels of disease burden-older adults whose disease burden had increased more over the past 20 years had higher base level of negative affect in their daily lives, more emotional reactivity to the experience of daily stressors, and more moment-to-moment fluctuations in negative affect that was unrelated to stressors (affective systemic noise). We highlight that developmental processes evolving over vastly different timescales are intertwined, and speculate how new knowledge about those relations can inform developmental theories of emotion regulation and daily-life functioning.
Public Significance Statement
How older adults master the challenges of their everyday lives is of utmost importance for their quality of life. Here, we draw from emotion regulation theories to examine if and how differences in affective dynamics of older adults' everyday life are related to differences in their accumulation of physical diseases over the prior two decades. Results revealed that-over and above current disease burden-the daily lives of older adults whose disease burden had increased more over the past 20 years are characterized by more negative affect, more emotional reactivity when faced with stressors, and more emotional lability. Our findings highlight that aspects of life that evolve at vastly different time metrics are linked and the need for future research on risk and protective factors that may moderate those links.
Over the past decade, many studies have reported individual differences in negative emotional reactions to daily stressful events. However, whether and how individual and age-related differences in ...emotional reactivity also depend on the temporal characteristics of stressors has received little attention. In this project, we focused on the temporal characteristics of stressor occurrence and examined the pile-up of stressors within a day-referring to multiple stressors encountered within a relatively narrow time window. To do so, we used data from 123 young-old (66-69 years, 47% women) and 47 very old adults (84-90 years, 60% women). Participants reported their momentary feelings and exposure to stressors six times a day over 7 consecutive days in their everyday life. Emotional reactivity to stressor pile-up over the day followed an exponential decay trajectory, with higher stressor burden in close proximity to the stressor occurrence. The exact shape of the decay trajectory differed among participants. Most importantly, both stressor pile-up and ongoing stress predicted greater emotional reactivity. We also found interaction effects of stressor pile-up and current stressor occurrence in that increases in negative affect under ongoing stress were stronger when stressors had piled-up before. No evidence was found for increased vulnerability to stressor pile-up in very old adults; rather, the impact of preceding stressors attenuated faster for individuals in this age group. The findings highlight the utility of comprehensively studying how stressor characteristics such as their pile-up within short time periods shape emotional reactivity.
This article examines the end-of-life development of depressive symptoms and characterizes prototypical groups following the same depressive symptoms development. We modeled time-to-death-related ...trajectories of depressive symptoms (Center for Epidemiologic Studies Depression Scale), applying a latent class growth analysis to deceased older adults from the English Longitudinal Study of Aging (Waves 1 to 5; NTime 1 (T1) = 2,219; MAge(T1) = 73.9 years, SDAge(T1) = 9.4 years; 51% male, 1% non-White). Four prototypical trajectories of depressive symptoms were identified at the end of life: a stably nondepressed group (31.2%); 2 groups with an exponential terminal symptom increase, of which 1 was nondepressed and 1 low depressed (8.3% and 38.4%, respectively); and a stably depressed group (22.2%). Using a combination of growth curve models and individual level and slope values as predictor variables showed that individuals suffering from increasing sensory, mobility, or overall health problems or decreasing quality of life were more likely to have an increase in symptoms of depression in their last years of life. Men were more likely to be stably nondepressed and women more likely to be chronically depressed. We conclude that a group-based analysis of end-of-life depressive symptoms is useful in adding to the understanding of distance-to-death psychology at large as well as pointing to preventive and intervention strategies when it comes to late-life depressive mood.
Adding to recent evidence of terminal decline in affective well-being, this study examined the role of physical health in accounting for time-to-death-related changes in positive affect (PA) and ...negative affect (NA). We distinguished effects of preterminal health levels predicting levels ("preserved differentiation") and terminal changes ("differential preservation") and of terminal health declines predicting terminal changes ("terminal reactivity") of affective well-being in the terminal period of life. Data were used from the first cohort of the Longitudinal Aging Study Amsterdam, including 3-yearly measurements from 1992-1993 to 2011-2012 (N = 2310, age = 55-85 at baseline). Measures of PA and NA were derived from the Center for Epidemiologic Studies Depression Scale. Health measures included self-rated health, self-reported functional limitations, and gait speed. Exponential time-to-death-related trajectories in PA and NA were analyzed with mixed regression models. Results confirmed accelerated time-to-death-related decline of PA and increase of NA. Regarding health effects, the findings support terminal reactivity, in that the amount of end-of-life changes in affective well-being was closely linked to the concurrent terminal changes in health. Preterminal health levels did not predict the amount of terminal changes in affective well-being; however, reaching the final years of life with better levels of functional health appeared to promote longer maintenance of better levels of affective well-being and terminal declines more "compressed" to a shorter period prior to death. The findings point to needs to strengthen individuals' resources to compensate for health losses at the end of their life span.
As proteome-wide C-terminal sequence analysis has been largely intractable, we developed a polymer-based enrichment approach to profile protein C-terminal peptides by mass spectrometry and identified ...hundreds of C-terminal peptides in the Escherichia coli proteome. We isotopically labeled GluC protease-digested and undigested samples and identified GluC substrates and their cleavage sites by quantification of neo-C-terminal peptides. Our method thus enables global annotation of protein C-terminal posttranslational modifications, including proteolytic truncations.
