Intrinsically disordered proteins (IDPs) are systematically under‐represented in structural proteomics studies. Their structural characterization implies description of the dynamic conformational ensembles populated by these polymers in solution, posing major challenges to biophysical methods. “Native” MS (native‐MS) has emerged as a central tool in this field, conjugating the unique MS analytical power with structurally meaningful descriptors, like solvent‐accessible surface area (SASA) and collisional cross section (CCS). This review summarizes recently published papers comparing native‐MS and solution methods, with a focus on charge‐state‐distribution (CSD) analysis for IDP conformational analysis. The results point to substantial agreement, supporting structural interpretation of native‐MS spectra of IDPs. The discussion is integrated with data from our group on “synthetic” IDPs, obtained by reduction and alkylation of natively folded proteins, whose fold is stabilized by disulfide bridges. Finally, an MS‐based compaction index (CI) is introduced, evaluating SASA with reference to globular and fully disorder proteins. Such a parameter can be calculated for single conformers or the whole conformational ensemble, offering a continuous index for IDP comparison and classification.

The lacrimal film has attracted increasing interest in the last decades as a potential source of biomarkers of physiopathological states, due to its accessibility, moderate complexity, and responsiveness to ocular and systemic diseases. High‐performance liquid chromatography‐mass spectrometry (LC‐MS) has led to effective approaches to tear proteomics, despite the intrinsic limitations in sample amounts. This review focuses on the recent progress in strategy and technology, with an emphasis on the potential for personalized medicine. After an introduction on lacrimal‐film composition, examples of applications to biomarker discovery are discussed, comparing approaches based on pooled‐sample and single‐tear analysis. Then, the most critical steps of the experimental pipeline, that is, tear collection, sample fractionation, and LC‐MS implementation, are discussed with reference to proteome‐coverage optimization. Advantages and challenges of the alternative procedures are highlighted. Despite the still limited number of studies, tear quantitative proteomics, including single‐tear investigation, could offer unique contributions to the identification of low‐invasiveness, sustained‐accessibility biomarkers, and to the development of personalized approaches to therapy and diagnosis.

PURPOSE. To evaluate the potential of lactoferrin (Lf) as a diagnostic biomarker for ocular diseases using a meta-analytic approach. METHODS. All original studies reporting an estimate of the average Lf concentration in healthy subjects and those affected by ocular diseases were searched up to March 2020. The DerSimonian and Laird method was used to calculate the random effects pooled mean difference and the corresponding 95% confidence interval (CI) in Lf concentration between healthy subjects and those affected by dry eye (DE), Sjögren syndrome (SS), and diabetic retinopathy, separately. The presence of between-study heterogeneity was evaluated using the Cochran's Q test and the I 2 index. Stratified analyses were performed to assess potential sources of heterogeneity and influence and cumulative analyses to evaluate the robustness of the results obtained. Publication bias was also evaluated using funnel plot and the Egger's test. RESULTS. The pooled mean differences in Lf concentrations between healthy subjects and those with DE, Sjögren syndrome, and diabetic retinopathy were respectively 0.62 (95% CI, 0.35-0.89) for DE, 3.78 (95% CI, −6.64 to 14.17), and 0.19 (95% CI, −4.00 to 4.39). Regarding DE, the stratified analysis showed that geographical area (P value Q test < 0.0001) and sample size (P < 0.0005) were sources of heterogeneity. Moreover, no study substantially influenced the results obtained and the pooled mean difference became statistically significant after a sample size of 220. Publication bias may affect the results of DE. CONCLUSIONS. The results of the current meta-analysis suggest that Lf level in tears is a good candidate as dry eye syndrome diagnostic biomarker.

Lacrimal fluid is an attractive source of noninvasive biomarkers, the main limitation being the small sample amounts typically collected. Advanced analytical methods to allow for proteomics profiling from a few microliters are needed to develop innovative biomarkers, with attractive perspectives of applications to precision medicine. This work describes an effective, analytical pipeline for single-tear analysis by ultrahigh-resolution, shotgun proteomics from 23 healthy human volunteers, leading to high-confidence identification of a total of 890 proteins. Highly reproducible quantification was achieved by either peak intensity, peak area, or spectral counting. Hierarchical clustering revealed a stratification of females vs. males that did not emerge from previous studies on pooled samples. Two subjects were monitored weekly over 3 weeks. The samples clustered by withdrawal time of day (morning vs. afternoon) but not by follow-up week, with elevated levels of components of the immune system in the morning samples. This study demonstrates feasibility of single-tear quantitative proteomics, envisaging contributions of this unconventional body fluid to individualized approaches in biomedicine.

Edited by Karen G. Fleming ␣-Synuclein (AS) is an intrinsically disordered protein highly expressed in dopaminergic neurons. Its amyloid aggregates are the major component of Lewy bodies, a hallmark of Parkinson's disease (PD). AS is particularly exposed to oxidation of its methionine residues, both in vivo and in vitro. Oxidative stress has been implicated in PD and oxidized ␣-synuclein has been shown to assemble into soluble, toxic oligomers, rather than amyloid fibrils. However, the structural effects of methionine oxidation are still poorly understood. In this work, oxidized AS was obtained by prolonged incubations with dopamine (DA) or epigallocatechin-3-gallate (EGCG), two inhibitors of AS aggregation, indicating that EGCG promotes the same final oxidation product as DA. The conformational transitions of the oxidized and non-oxidized protein were monitored by complementary biophysical techniques, including MS, ion mobility (IM), CD, and FTIR spectroscopy assays. Although the two variants displayed very similar structures under conditions that stabilize highly disordered or highly ordered states, differences emerged in the intermediate points of transitions induced by organic solvents, such as trifluoroethanol (TFE) and methanol (MeOH), indicating a lower propensity of the oxidized protein for forming either ␣or ␤-type secondary structures. Furthermore, oxidized AS displayed restricted secondary-structure transitions in response to dehydration and slightly amplified tertiary-structure transitions induced by ligand binding. This difference in susceptibility to induced folding could explain the loss of fibrillation potential observed for oxidized AS. Finally, site-specific oxidation kinetics point out a minor delay in Met-127 modification, likely due to the effects of AS intrinsic structure.