Robust assessment of sample preparation protocols for proteomics of cells and tissues.

In proteomic studies, the reliability and reproducibility of results hinge on well-executed protein extraction and digestion protocols. Here, we systematically compared three established digestion methods for macrophages, namely filter-assisted sample preparation (FASP), in-solution, and in-gel digestion protocols. We also compared lyophilization and manual lysis for liver tissue protein extraction, each of them tested using either sodium deoxycholate (SDC)- or RIPA-based lysis buffer. For the macrophage cell line, FASP using passivated filter units outperformed the other tested methods regarding the number of identified peptides and proteins. However, a careful standardization has shown that all three methods can yield robust results across a wide range of starting material (even starting with 1 µg of proteins). Importantly, inter and intra-day coefficients of variance (CVs) were determined for all sample preparation protocols. Thus, the median inter-day CVs for in-solution, in-gel and FASP protocols were respectively 10, 8 and 9%, very similar to the median CVs obtained for the intra-day analysis (9, 8 and 8%, respectively). Moreover, FASP digestion presented 80% of proteins with a CV lower than 25%, followed closely by in-gel digestion (78%) and in-solution sample preparation (72%) protocols. For tissue proteomics, both manual lysis and lyophilization presented similar proteome coverage and reproducibility, but the efficiency of protein extraction depended on the lysis buffer used, with RIPA buffer showing better results. In conclusion, although each sample preparation method has its own particularity, they are all suited for successful proteomic experiments if a careful standardization of the sample preparation workflow is carried out.

Glucose restriction in Saccharomyces cerevisiae modulates the phosphorylation pattern of the 20S proteasome and increases its activity.

Caloric restriction is known to extend the lifespan and/or improve diverse physiological parameters in a vast array of organisms. In the yeast Saccharomyces cerevisiae, caloric restriction is performed by reducing the glucose concentration in the culture medium, a condition previously associated with increased chronological lifespan and 20S proteasome activity in cell extracts, which was not due to increased proteasome amounts in restricted cells. Herein, we sought to investigate the mechanisms through which glucose restriction improved proteasome activity and whether these activity changes were associated with modifications in the particle conformation. We show that glucose restriction increases the ability of 20S proteasomes, isolated from Saccharomyces cerevisiae cells, to degrade model substrates and whole proteins. In addition, threonine 55 and/or serine 56 of the α5-subunit, were/was consistently found to be phosphorylated in proteasomes isolated from glucose restricted cells, which may be involved in the increased proteolysis capacity of proteasomes from restricted cells. We were not able to observe changes in the gate opening nor in the spatial conformation in 20S proteasome particles isolated from glucose restricted cells, suggesting that the changes in activity were not accompanied by large conformational alterations in the 20S proteasome but involved allosteric activation of proteasome catalytic site.

Strategies for consistent and automated quantification of HDL proteome using data-independent acquisition.

The introduction of mass spectrometry-based proteomics has revolutionized the high-density lipoprotein (HDL) field, with the description, characterization, and implication of HDL-associated proteins in an array of pathologies. However, acquiring robust, reproducible data is still a challenge in the quantitative assessment of HDL proteome. Data-independent acquisition (DIA) is a mass spectrometry methodology that allows the acquisition of reproducible data, but data analysis remains a challenge in the field. To date, there is no consensus on how to process DIA-derived data for HDL proteomics. Here, we developed a pipeline aiming to standardize HDL proteome quantification. We optimized instrument parameters and compared the performance of four freely available, user-friendly software tools (DIA-NN, EncyclopeDIA, MaxDIA, and Skyline) in processing DIA data. Importantly, pooled samples were used as quality controls throughout our experimental setup. A careful evaluation of precision, linearity, and detection limits, first using E. coli background for HDL proteomics and second using HDL proteome and synthetic peptides, was undertaken. Finally, as a proof of concept, we employed our optimized and automated pipeline to quantify the proteome of HDL and apolipoprotein B-containing lipoproteins. Our results show that determination of precision is key to confidently and consistently quantifying HDL proteins. Taking this precaution, any of the available software tested here would be appropriate for quantification of HDL proteome, although their performance varied considerably.

Citrullination of actin-ligand and nuclear structural proteins, cytoskeleton reorganization and protein redistribution across cellular fractions are early events in ionomycin-induced NETosis.

Neutrophil extracellular traps (NETs) are web-like structures of DNA coated with cytotoxic proteins and histones released by activated neutrophils through a process called NETosis. NETs release occurs through a sequence of highly organized events leading to chromatin expansion and rupture of nuclear and cellular membranes. In calcium ionophore-induced NETosis, the enzyme peptidylargine deiminase 4 (PAD4) mediates chromatin decondensation through histone citrullination, but the biochemical pathways involved in this process are not fully understood. Here we use live-imaging microscopy and proteomic studies of the neutrophil cellular fractions to investigate the early events in ionomycin-triggered NETosis. We found that before ionomycin-stimulated neutrophils release NETs, profound biochemical changes occur in and around their nucleus, such as, cytoskeleton reorganization, nuclear redistribution of actin-remodeling related proteins, and citrullination of actin-ligand and nuclear structural proteins. Ionomycin-stimulated neutrophils rapidly lose their characteristic polymorphic nucleus, and these changes are promptly communicated to the extracellular environment through the secretion of proteins related to immune response. Therefore, our findings revealed key biochemical mediators in the early process that subsequently culminates with nuclear and cell membranes rupture, and extracellular DNA release.

Singlet Molecular Oxygen Generation in the Reaction of Biological Haloamines of Amino Acids and Polyamines with Hydrogen Peroxide†.

Leucocytes generate hypohalous acids (HOCl and HOBr) to defend the host against pathogens. In cells, hypohalous acids react with amine-containing molecules, such as amino acids and polyamines, producing chloramines and bromamines, reservoirs of oxidizing power that can potentially damage host tissues at sites of inflammation. Hypohalous acids also react with H2 O2 to produce stoichiometric amounts of singlet molecular oxygen ( 1 O 2 ), but its generation in leucocytes is still under debate. Additionally, it is unclear whether haloamines generate 1 O 2 following a reaction with H2 O2 . Herein, we provide evidence of the generation of 1 O 2 in the reactions between amino acid-derived (taurine, N-α-acetyl-Lysine and glycine) and polyamine-derived (spermine and spermidine) haloamines and H2 O2 in an aqueous solution. The unequivocal formation of 1 O 2 was detected by monitoring its characteristic monomol light emission at 1270 nm in the near-infrared region. For amino acid-derived haloamines, the presence of 1 O 2 was further confirmed by chemical trapping with anthracene-9,10-divinylsulfonate and HPLC-MS/MS detection. Altogether, photoemission and chemical trapping studies demonstrated that chloramines were less effective at producing 1 O 2 than bromamines of amino acids and polyamines. Thus, 1 O 2 formation via bromamines and H2 O2 may be a potential source of 1 O 2 in nonilluminated biological systems.

HDL Isolated by Immunoaffinity, Ultracentrifugation, or Precipitation is Compositionally and Functionally Distinct.

The HDL proteome has been widely recognized as an important mediator of HDL function. While a variety of HDL isolation methods exist, their impact on the HDL proteome and its associated function remain largely unknown. Here, we compared three of the most common methods for HDL isolation, namely immunoaffinity (IA), density gradient ultracentrifugation (UC), and dextran-sulfate precipitation (DS), in terms of their effects on the HDL proteome and associated functionalities. We used state-of-the-art mass spectrometry to identify 171 proteins across all three isolation methods. IA-HDL contained higher levels of paraoxonase 1, apoB, clusterin, vitronectin, and fibronectin, while UC-HDL had higher levels of apoA2, apoC3, and α-1-antytrypsin. DS-HDL was enriched with apoA4 and complement proteins, while the apoA2 content was very low. Importantly, size-exclusion chromatography analysis showed that IA-HDL isolates contained subspecies in the size range above 12 nm, which were entirely absent in UC-HDL and DS-HDL isolates. Analysis of these subspecies indicated that they primarily consisted of apoA1, IGκC, apoC1, and clusterin. Functional analysis revealed that paraoxonase 1 activity was almost completely lost in IA-HDL, despite high paraoxonase content. We observed that the elution conditions, using 3M thiocyanate, during IA resulted in an almost complete loss of paraoxonase 1 activity. Notably, the cholesterol efflux capacity of UC-HDL and DS-HDL was significantly higher compared to IA-HDL. Together, our data clearly demonstrate that the isolation procedure has a substantial impact on the composition, subclass distribution, and functionality of HDL. In summary, our data show that the isolation procedure has a significant impact on the composition, subclass distribution and functionality of HDL. Our data can be helpful in the comparison, replication and analysis of proteomic datasets of HDL.

Dehydromethionine is a common product of methionine oxidation by singlet molecular oxygen and hypohalous acids.

Methionine is one of the main targets for biological oxidants. Its reaction with the majority of oxidants generates only methionine sulfoxide. However, when N-terminal methionine reacts with hypohalous acids (HOCl and HOBr) or singlet molecular oxygen (1O2), it can also generate a cyclic product called dehydromethionine (DHM). Previously, DHM was suggested as a biomarker of oxidative stress induced by hypohalous acids. However, DHM can also be generated by 1O2 -oxidation of methionine, and the contribution of this pathway of DHM formation in a context of a site-specific redox imbalance in an organism is unknown. In this work, a through comparison of the reactions of hypohalous acids and 1O2 with methionine, either free or inserted in peptides and proteins was undertaken. In addition, we performed methionine photooxidation in heavy water (H218O) to determine the influence of the pH in the mechanism of DHM formation. We showed that for free methionine, or methionine-containing peptides, the yields of DHM formation in the reactions with 1O2 were close to those achieved by HOBr oxidation, but much higher than the yields obtained with HOCl as the oxidant. This was true for all pH tested (5, 7.4, and 9). Interestingly, for the protein ubiquitin, DHM yields after reaction with 1O2 were higher than those obtained with both hypohalous acids. Our results indicate that 1O2 may also be an important source of DHM in biological systems.

Spatial proteomics reveals subcellular reorganization in human keratinocytes exposed to UVA light.

The effects of UV light on the skin have been extensively investigated. However, systematic information about how the exposure to ultraviolet-A (UVA) light, the least energetic but the most abundant UV radiation reaching the Earth, shapes the subcellular organization of proteins is lacking. Using subcellular fractionation, mass-spectrometry-based proteomics, machine learning algorithms, immunofluorescence, and functional assays, we mapped the subcellular reorganization of the proteome of human keratinocytes in response to UVA light. Our workflow quantified and assigned subcellular localization for over 1,600 proteins, of which about 200 were found to redistribute upon UVA exposure. Reorganization of the proteome affected modulators of signaling pathways, cellular metabolism, and DNA damage response. Strikingly, mitochondria were identified as one of the main targets of UVA-induced stress. Further investigation demonstrated that UVA induces mitochondrial fragmentation, up-regulates redox-responsive proteins, and attenuates respiratory rates. These observations emphasize the role of this radiation as a potent metabolic stressor in the skin.

Characterization and Quantification of Tryptophan- and Tyrosine-derived Hydroperoxides.

The reaction of singlet oxygen (1 O2 ) with the amino acids tryptophan and tyrosine, either free or inserted in peptides or proteins, gives rise to hydroperoxides. To understand the impact of these hydroperoxides in complex biological systems, methods allowing their characterization and accurate quantification must be available. In this work, hydroperoxides derived from tryptophan and tyrosine and from peptides containing these amino acids were synthesized by photooxidation, and characterized by high-resolution mass spectrometry. In addition, experiments were carried out to compare two colorimetric methods commonly used for quantification of peroxides, namely the iodometric and the ferric-xylenol orange assays. For the tryptophan hydroperoxide, the quantifications obtained by colorimetric methods were then compared to that obtained by NMR. The results showed that for the ferric-xylenol orange method, the stoichiometry between peroxide and Fe3+ ions varies considerably. On the other hand, for the iodometric assay, the stoichiometry peroxide:I3 - ions is always 1:1. However, the kinetics of the reactions of peroxides with I- vary, and the assay must be performed in anaerobic conditions. Thus, the iodometric method is more appropriate for precise quantification of a given peroxide. The characterization and accurate quantification of biological peroxides is key to understand the mechanisms involved in redox processes.

A single dose of Ultraviolet-A induces proteome remodeling and senescence in primary human keratinocytes.

Epidermal photoaging contributes to skin fragility over time and it is a risk factor for skin cancer. Photoaging has been associated for a long time with exposure to Ultraviolet-A (UVA) light, the predominant component of the solar ultraviolet radiation. While the cellular mechanisms underlying UVA-induced photoaging in the dermis have been well characterized, UVA's action on the epidermis remains elusive. Here, proteomic analysis was conducted to derive the cellular responses induced by an environmentally relevant dose of UVA in primary human keratinocytes. We also investigated the effects of UVA on non-transformed immortalized keratinocytes (HaCaT cells), bearing potentially oncogenic mutations. We showed that UVA induces proteome remodeling and senescence in primary keratinocytes, eliciting potent antioxidant and pro-inflammatory responses. Additionally, we showed that UVA modulates the secretory phenotype of these cells to the extent of inducing paracrine oxidative stress and immune system activation in pre-malignant keratinocytes. These observations offer insights into the cellular mechanisms by which UVA drives photoaging in the skin.

HDL proteome remodeling associates with COVID-19 severity.

BACKGROUND: Besides the well-accepted role in lipid metabolism, high-density lipoprotein (HDL) also seems to participate in host immune response against infectious diseases. OBJECTIVE: We used a quantitative proteomic approach to test the hypothesis that alterations in HDL proteome associate with severity of Coronavirus disease 2019 (COVID-19). METHODS: Based on clinical criteria, subjects (n=41) diagnosed with COVID-19 were divided into two groups: a group of subjects presenting mild symptoms and a second group displaying severe symptoms and requiring hospitalization. Using a proteomic approach, we quantified the levels of 29 proteins in HDL particles derived from these subjects. RESULTS: We showed that the levels of serum amyloid A 1 and 2 (SAA1 and SAA2, respectively), pulmonary surfactant-associated protein B (SFTPB), apolipoprotein F (APOF), and inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) were increased by more than 50% in hospitalized patients, independently of sex, HDL-C or triglycerides when comparing with subjects presenting only mild symptoms. Altered HDL proteins were able to classify COVID-19 subjects according to the severity of the disease (error rate 4.9%). Moreover, apolipoprotein M (APOM) in HDL was inversely associated with odds of death due to COVID-19 complications (odds ratio [OR] per 1-SD increase in APOM was 0.27, with 95% confidence interval [CI] of 0.07 to 0.72, P=0.007). CONCLUSION: Our results point to a profound inflammatory remodeling of HDL proteome tracking with severity of COVID-19 infection. They also raise the possibility that HDL particles could play an important role in infectious diseases.

Human cataractous lenses contain cross-links produced by crystallin-derived tryptophanyl and tyrosyl radicals.

Protein insolubilization, cross-linking and aggregation are considered critical to the development of lens opacity in cataract. However, the information about the presence of cross-links other than disulfides in cataractous lenses is limited. A potential role for cross-links produced from tryptophanyl radicals in cataract development is suggested by the abundance of the UV light-sensitive Trp residues in crystallin proteins. Here we developed a LC-MS/MS approach to examine the presence of Trp-Trp, Trp-Tyr and Tyr-Tyr cross-links and of peptides containing Trp-2H (-2.0156 Da) in the lens of three patients diagnosed with advanced nuclear cataract. In the proteins of two of the lenses, we characterized intermolecular cross-links between ßB2-Tyr153-Tyr104-ßA3 and ßB2-Trp150-Tyr139-ßS. An additional intermolecular cross-link (ßB2-Tyr61-Trp200-ßB3) was present in the lens of the oldest patient. In the proteins of all three lenses, we characterized two intramolecular Trp-Trp cross-links (Trp123-Trp126 in ßB1 and Trp81-Trp84 in ßB2) and six peptides containing Trp -2H residues, which indicate the presence of additional Trp-Trp cross-links. Relevantly, we showed that similar cross-links and peptides with modified Trp-2H residues are produced in a time-dependent manner in bovine ß-crystallin irradiated with a solar simulator. Therefore, different crystallin proteins cross-linked by crystalline-derived tryptophanyl and tyrosyl radicals are present in advanced nuclear cataract lenses and similar protein modifications can be promoted by solar irradiation even in the absence of photosensitizers. Overall, the results indicate that a role for Trp-Tyr and Trp-Trp cross-links in the development of human cataract is possible and deserves further investigation.

Deepening our understanding of HDL proteome.

Introduction: High-density lipoprotein (HDL) particles are heterogeneous and their proteome is complex and distinct from HDL cholesterol. However, it is largely unknown whether HDL proteins are associated with cardiovascular protection. Areas covered: HDL isolation techniques and proteomic analyses are reviewed. A list of HDL proteins reported in 37 different studies was compiled and the effects of different isolation techniques on proteins attributed to HDL are discussed. Mass spectrometric techniques used for HDL analysis and the need for precise and robust methods for quantification of HDL proteins are discussed. Expert opinion: Proteins associated with HDL have the potential to be used as biomarkers and/or help to understand HDL functionality. To achieve this, large cohorts must be studied using precise quantification methods. Key factors in HDL proteome quantification are the isolation methodology and the mass spectrometry technique employed. Isolation methodology affects what proteins are identified in HDL and the specificity of association with HDL particles needs to be addressed. Shotgun proteomics yields imprecise quantification, but the majority of HDL studies relied on this approach. Few recent studies used targeted tandem mass spectrometry to quantify HDL proteins, and it is imperative that future studies focus on the application of these precise techniques.

Mechanism of dioxindolylalanine formation by singlet molecular oxygen-mediated oxidation of tryptophan residues.

The singlet molecular oxygen-oxidation of tryptophan generates diastereoisomeric dioxindolylalanine (diOia) along with hydroperoxides, alcohols and carbonyl compounds. Mechanistic investigations based on isotopic labeling and MS/MS analyses support diOia formation through a dioxetane intermediate.

Characterization of O(2) ((1)delta(g))-derived oxidation products of tryptophan: a combination of tandem mass spectrometry analyses and isotopic labeling studies.

The fragmentation mechanisms of singlet oxygen [O(2) ((1)Delta(g))]-derived oxidation products of tryptophan (W) were analyzed using collision-induced dissociation coupled with (18)O-isotopic labeling experiments and accurate mass measurements. The five identified oxidized products, namely two isomeric alcohols (trans and cis WOH), two isomeric hydroperoxides (trans and cis WOOH), and N-formylkynurenine (FMK), were shown to share some common fragment ions and losses of small neutral molecules. Conversely, each oxidation product has its own fragmentation mechanism and intermediates, which were confirmed by (18)O-labeling studies. Isomeric WOH lost mainly H(2)O + CO, while WOOH showed preferential elimination of C(2)H(5)NO(3) by two distinct mechanisms. Differences in the spatial arrangement of the two isomeric WOHs led to differences in the intensities of the fragment ions. The same behavior was also found for trans and cis WOOH. FMK was shown to dissociate by a diverse range of mechanisms, with the loss of ammonia the most favored route. MS/MS analyses, (18)O-labeling, and H(2)(18)O experiments demonstrated the ability of FMK to exchange its oxygen atoms with water. Moreover, this approach also revealed that the carbonyl group has more pronounced oxygen exchange ability compared with the formyl group. The understanding of fragmentation mechanisms involved in O(2) ((1)Delta(g))-mediated oxidation of W provides a useful step toward the structural characterization of oxidized peptides and proteins.

Cytoprotective effects of carvedilol against oxygen free radical generation in rat liver.

The protective effects of carvedilol, an antihypertensive agent, against oxidative injury caused by acetaminophen were studied in rat liver. Male Wistar rats (250 +/- 30 g) were pre-treated with carvedilol (3.6 mg/kg, p.o.) for 10 days and on the 11th day received an overdose of acetaminophen (800 mg/kg, p.o.). Four hours after acetaminophen administration, blood was collected to determine serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT). After that, rats were killed and the livers were excised to determine reduced glutathione (GSH), thiobarbituric acid reactive substances (TBARS) and carbonyl protein contents, and the activity of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione S-transferase (GST), and also the DNA damage index. Acetaminophen significantly increased the levels of TBARS, the DNA damage and SOD, AST and ALT activities. Carvedilol was able to prevent lipid peroxidation, protein carbonilation and DNA fragmentation caused by acetaminophen. Moreover, this drug prevented increases in SOD, AST and ALT activities. These results show that carvedilol exerts cytoprotective effects against oxidative injury caused by acetaminophen in rat liver. These effects are probably related to the O2*- scavenging property of carvedilol or its metabolites.