Cellular Redox Systems Impact the Aggregation of Cu,Zn Superoxide Dismutase Linked to Familial Amyotrophic Lateral Sclerosis.

Protein misfolding is implicated in neurodegenerative diseases such as ALS, where mutations of superoxide dismutase 1 (SOD1) account for about 20% of the inherited mutations. Human SOD1 (hSOD1) contains four cysteines, including Cys(57) and Cys(146), which have been linked to protein stability and folding via forming a disulfide bond, and Cys(6) and Cys(111) as free thiols. But the roles of the cellular oxidation-reduction (redox) environment in SOD1 folding and aggregation are not well understood. Here we explore the effects of cellular redox systems on the aggregation of hSOD1 proteins. We found that the known hSOD1 mutations G93A and A4V increased the capability of the thioredoxin and glutaredoxin systems to reduce hSOD1 compared with wild-type hSOD1. Treatment with inhibitors of these redox systems resulted in an increase of hSOD1 aggregates in the cytoplasm of cells transfected with mutants but not in cells transfected with wild-type hSOD1 or those containing a secondary C111G mutation. This aggregation may be coupled to changes in the redox state of the G93A and A4V mutants upon mild oxidative stress. These results strongly suggest that the thioredoxin and glutaredoxin systems are the key regulators for hSOD1 aggregation and may play critical roles in the pathogenesis of ALS.

In silico instrumental response correction improves precision of label-free proteomics and accuracy of proteomics-based predictive models.

In the analysis of proteome changes arising during the early stages of a biological process (e.g. disease or drug treatment) or from the indirect influence of an important factor, the biological variations of interest are often small (∼10%). The corresponding requirements for the precision of proteomics analysis are high, and this often poses a challenge, especially when employing label-free quantification. One of the main contributors to the inaccuracy of label-free proteomics experiments is the variability of the instrumental response during LC-MS/MS runs. Such variability might include fluctuations in the electrospray current, transmission efficiency from the air-vacuum interface to the detector, and detection sensitivity. We have developed an in silico post-processing method of reducing these variations, and have thus significantly improved the precision of label-free proteomics analysis. For abundant blood plasma proteins, a coefficient of variation of approximately 1% was achieved, which allowed for sex differentiation in pooled samples and ≈90% accurate differentiation of individual samples by means of a single LC-MS/MS analysis. This method improves the precision of measurements and increases the accuracy of predictive models based on the measurements. The post-acquisition nature of the correction technique and its generality promise its widespread application in LC-MS/MS-based methods such as proteomics and metabolomics.

Predictive urinary biomarkers for steroid-resistant and steroid-sensitive focal segmental glomerulosclerosis using high resolution mass spectrometry and multivariate statistical analysis.

BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is a glomerular scarring disease diagnosed mostly by kidney biopsy. Since there is currently no diagnostic test that can accurately predict steroid responsiveness in FSGS, prediction of the responsiveness of patients to steroid therapy with noninvasive means has become a critical issue. In the present study urinary proteomics was used as a noninvasive tool to discover potential predictive biomarkers. METHODS: Urinary proteome of 10 patients (n = 6 steroid-sensitive, n = 4 steroid-resistant) with biopsy proven FSGS was analyzed using nano-LC-MS/MS and supervised multivariate statistical analysis was performed. RESULTS: Twenty one proteins were identified as discriminating species among which apolipoprotein A-1 and Matrix-remodeling protein 8 had the most drastic fold changes being over- and underrepresented, respectively, in steroid sensitive compared to steroid resistant urine samples. Gene ontology enrichment analysis revealed acute inflammatory response as the dominant biological process. CONCLUSION: The obtained results suggest a panel of predictive biomarkers for FSGS. Proteins involved in the inflammatory response are shown to be implicated in the responsiveness. As a tool for biomarker discovery, urinary proteomics is especially fruitful in the area of prediction of responsiveness to drugs. Further validation of these biomarkers is however needed.

Brain proteomics supports the role of glutamate metabolism and suggests other metabolic alterations in protein l-isoaspartyl methyltransferase (PIMT)-knockout mice.

Protein l-isoaspartyl methyltransferase (PIMT) repairs the isoaspartyl residues (isoAsp) that originate from asparagine deamidation and aspartic acid (Asp) isomerization to Asp residues. Deletion of the gene encoding PIMT in mice (Pcmt1) leads to isoAsp accumulation in all tissues measured, especially in the brain. These PIMT-knockout (PIMT-KO) mice have perturbed glutamate metabolism and die prematurely of epileptic seizures. To elucidate the role of PIMT further, brain proteomes of PIMT-KO mice and controls were analyzed. The isoAsp levels from two of the detected 67 isoAsp sites (residue 98 from calmodulin and 68 from glyceraldehyde-3-phosphate dehydrogenase) were quantified and found to be significantly increased in PIMT-KO mice (p < 0.01). Additionally, the abundance of at least 151 out of the 1017 quantified proteins was found to be altered in PIMT-KO mouse brains. Gene ontology analysis revealed that many down-regulated proteins are involved in cellular amino acid biosynthesis. For example, the serine synthesis pathway was suppressed, possibly leading to reduced serine production in PIMT-KO mice. Additionally, the abundances of enzymes in the glutamate-glutamine cycle were altered toward the accumulation of glutamate. These findings support the involvement of PIMT in glutamate metabolism and suggest that the absence of PIMT also affects other processes involving amino acid synthesis and metabolism.

Mass spectrometric analysis of asparagine deamidation and aspartate isomerization in polypeptides.

One of the most frequent modifications in proteins and peptides is the deamidation of asparagine, a spontaneous non-enzymatic reaction leading to a mixture of L,D-succinimidyl, L,D-aspartyl, and L,D-isoaspartyl forms, with L-isoaspartyl dominating. Spontaneous isomerization of L-Asp yields the same products. In vivo, these unusual forms of aspartate are repaired by the protein L-isoaspartyl O-methyltransferase enzyme, with the balance between isomerization and repair affecting the organism physiology. Mass spectrometric analysis of this balance involves isomer separation, iso-Asp/Asp quantification, and iso-Asp site identification. This review highlights the issues associated with these steps and discusses the prospects of high-throughput iso-Asp analysis.

Toward proteome-scale identification and quantification of isoaspartyl residues in biological samples.

Deamidation of asparaginyl and isomerization of aspartyl residues in proteins produce a mixture of aspartyl and isoaspartyl residues, the latter being involved in protein aging and inactivation. Electron capture dissociation (ECD) combined with Fourier transform mass spectrometry (FT MS) are known to be able to distinguish the isoaspartyl peptides by unique fragments of cn* + 58.0054 (C2H2O2) and z(l-n)-56.9976 (C2HO2), where n is the position of the aspartyl residue and l is the peptide length. In the present study, we tested the specificity of isoAsp detection using the accurate masses of the specific fragments. For this purpose, we analyzed 32 whole and partial proteomes obtained from human cells as well as tissue samples and identified by ECD 466 isoaspartyl peptide candidates. Detailed inspection revealed that many of these candidates were unreliable. To increase the isoAsp detection specificity, additional criteria had to be used, for example, adjacent c/z fragments, specific losses from the reduced species, and the shape of the chromatographic peak. Most stringent filtering of candidates yielded several cases where the presence of isoAsp was beyond doubt. Among the identified proteins with isoAsp, actin, heat shock cognate 71 kDa protein and pyruvate kinase have previously been identified as substrates for l-isoaspartyl methyltransferase, an important repair enzyme converting isoaspartyl to aspartyl. Quantification of relative isomerization degree was performed by the label-free approach. This is the first attempt to analyze the human isoaspartome in a high-throughput manner. The developed workflow allows for further enhancement of the detection rate of isoaspartyl residues in biological samples.

Prognostic polypeptide blood plasma biomarkers of Alzheimer's disease progression.

BACKGROUND: Patients with mild cognitive impairment (MCI) have varying risks of progression to Alzheimer's disease (AD). OBJECTIVE: To test the utility of the relative abundances of blood plasma polypeptides for predicting the risk of AD progression. METHODS: 119 blood plasma samples of patients with MCI with different outcomes (stable MCI and progressive MCI) were analyzed by untargeted, label-free shotgun proteomics. Predictive biomarkers of progressive MCI were selected by multivariate analysis, followed by cross-validation of the predictive model. RESULTS: The best model demonstrated the accuracy of ca. 79% in predicting progressive MCI. Sex differences of the predictive biomarkers were also assessed. We have identified some sex-specific protein biomarkers, e.g., alpha-2-macrogloblin (A2M), which strongly correlates with female AD progression but not with males. CONCLUSION: Significant sex bias in AD-specific biomarkers underscores the necessity of selecting sex-balanced cohort in AD biomarker studies, or using sex-specific models. Blood protein biomarkers are found to be promising for predicting AD progression in clinical settings.

Blood plasma IgG Fc glycans are significantly altered in Alzheimer's disease and progressive mild cognitive impairment.

Blood-based anti-amyloid-ß (Aß) immunoglobulins (IgGs) and peripheral inflammation are factors correlating with development of Alzheimer's disease (AD). IgG functionality can drastically change from anti- to pro-inflammatory via alterations in the IgG-Fc N-glycan structure. Herein, we tested if IgG-Fc glycosylation in plasma is indeed altered during the development of AD. Samples from age-matched subjects of 23 controls, 58 patients with stable mild cognitive impairment (SMCI), 34 patients with progressive (P)MCI, and 31 patients with AD were investigated. Label-free shotgun proteomics was applied without glycoprotein enrichment. Glycans on peptides EEQYNSTYR (IgG1) and EEQFNSTFR (IgG2) were quantified, and their abundances were normalized to total IgGn glycoform abundance. Univariate and multivariate statistics were employed to investigate the correlations between the patients groups and the abundances of the IgG glycoforms as well as those of inflammatory mediating proteins. Significant differences (p ≤ 0.05) were found, with a lower abundance of complex galactosylated and sialylated forms in AD. For females, a decline in glycoform complexity correlated with disease progress but an inverse change was found in males prior to the onset of AD. Principal component analysis (PCA; Males: R(2)X(cum) = 0.65, Q(2)(cum) = 0.34; Females: R(2)X(cum) = 0.62, Q(2)(cum) = 0.36), confirmed the gender similarities (for controls, SMCI and AD) as well as differences (for PMCI), and showed a close correlation between pro-inflammatory protein markers, AD, female PMCI, and truncated IgG-Fc glycans. The differences observed between genders prior to the onset of AD may indicate a lower ability in females to suppress peripheral inflammation, which may lead to exacerbated disease progression.

N-terminal peptide sequence repetition influences the kinetics of backbone fragmentation: a manifestation of the Jahn-Teller effect?

Analysis of large (>10,000 entries) databases consisting of high-resolution tandem mass spectra of peptide dications revealed with high statistical significance (P < 1[Symbol: see text]10(-3)) that peptides with non-identical first two N-terminal amino acids undergo cleavages of the second peptide bond at higher rates than repetitive sequences composed of the same amino acids (i.e., in general AB- and BA- bonds cleave more often than AA- and BB- bonds). This effect seems to depend upon the collisional energy, being stronger at lower energies. The phenomenon is likely to indicate the presence of the diketopiperazine structure for at least some b2 (+) ions. When consisting of two identical amino acids, these species should form through intermediates that have a symmetric geometry and, thus, must be subject to the Jahn-Teller effect that reduces the stability of such systems.

Shotgun brain proteomics reveals early molecular signature in presymptomatic mouse model of Alzheimer's disease.

AßpE3-42 (N-terminal truncated amyloid-ß peptide starting with pyroglutamate at the third position) is abundant in Alzheimer's disease (AD) brain and has high aggregation propensity and cellular toxicity. Transgenic TBA42 mice expressing AßpE3-42 exhibit a neurological phenotype evident at the age of 12 months. As AD has a long presymptomatic period, early detection of imminent neurodegeneration is highly desirable. In the present work we used four-month-old presymptomatic TBA42 mice and performed a whole-brain proteome analysis in order to elucidate early AD-related pathological changes and the molecular networks involved. At least three proteins were found to be moderately (by 17% to 28%) but statistically significantly upregulated, including: nectin-like molecule 1 involved in cell-cell adhesion; Homer proteins involved in scaffolding, organizing proteins at synapse and regulating intracellular calcium within neurons; and inositol-trisphosphate 3-kinase A, which is important for InsP3 induced calcium signaling in the brain. Analysis of key nodes (regulatory molecules found on pathway intersections) identified Rho-kinase (ROCK), a serine/threonine kinase and one of the major downstream effectors of the small GTPase Rho, as well as three key nodes of the mTOR/p70S6K signaling pathway previously implicated in multiple fundamental biological processes including synaptic plasticity, and upregulated in AD. These data confirm that AD-typical molecular pathways can be detected by whole-brain shotgun proteomics in young presymptomatic mice long before the onset of behavioral changes.

Carbonyl charge solvation patterns may relate to fragmentation classes in collision-activated dissociation.

Here, we investigate the hypothesis that the origin of Class I fragmentation in tryptic peptide dications corresponding to the cleavage of the first two amino acids from the N-terminus is due to a dominant charge solvation pattern. Molecular dynamics simulations (MDS) of model A(n)R dications confirmed the existence of a persistent solvation of the protonated N-terminus on the second backbone carbonyl. Additionally, MDS predicted a new distinct fragmentation class corresponding to the loss of two amino acids from the C-terminus. This prediction was confirmed experimentally at very low excitation levels. The pattern produced by electron transfer dissociation of the same dications gave markedly decreased cleavage frequencies at the second peptide bond, which, within the non-local fragmentation mechanism, supports the preferential charge solvation on the second carbonyl. Taken together, these results confirm the role of a charge solvation pattern in the origin of fragmentation classes.

Introducing an Asp-Pro linker in the synthesis of random one-bead-one-compound hexapeptide libraries compatible with ESI-MS analysis.

A random hexapeptide library (one-bead-one-compound), containing sixteen amino acids (16(6) different sequences) was synthesized on a Tentagel resin previously modified with a dipeptide linker (Asp-Pro). This peptide bond is highly susceptible to cleavage under mild acidic conditions in a salt-free solution prepared with H(2)(16)O/H(2)(18)O (60/40% v/v). In the hydrolysis, hexapeptides are released with an additional Asp residue partially labeled with (18)O at the C-terminus. These conditions are fully compatible with ESI-MS analysis and facilitate sequencing by MS, as N- and C-terminal ions can be easily differentiated in MS/MS spectra. The peptides were sequenced manually and also with de novo sequencing programs, and identifying them in a database containing all possible heptapeptide sequences or in a filtered database. The proposed strategy is also compatible with stepwise Edman degradation using either intact beads or the released free peptides.

Alzheimer's disease and mild cognitive impairment are associated with elevated levels of isoaspartyl residues in blood plasma proteins.

Increased levels of isoaspartyl residues (isoAsp) have previously been found in proteins of Alzheimer's disease (AD) brains and in blood proteins of patients suffering from uremia, the disease sharing common pathological features with AD. One can hypothesize that higher levels of isoAsp should be present in blood proteins of AD patients. Also, because of higher AD prevalence in females, they can be expected to have higher level of isoAsp than males. Here we modified our recently developed proteome-wide isoAsp analysis approach for testing these hypotheses. Eight blood plasma samples pooled from 218 individuals suffering from either mild cognitive impairment (MCI) or AD were analyzed by tandem mass spectrometry using electron transfer dissociation. Based on specific fragmentation pattern of isoAsp, the healthy controls were found to contain lower level of isoAsp compared with age-matched MCI and AD patients (p = 0.03). This result was further validated (p = 0.05) by 96 individual sample analyses, giving the combined value of p ≈ 0.01. Female pooled samples were found to contain higher level of isoAsp than male in both pooled and individual samples, with overall p ≈ 0.01. These findings verify the above hypotheses, and provide protein candidates for further investigation of the link between isoAsp and AD.

Heme binding in gas-phase holo-myoglobin cations: distal becomes proximal?

His64 and His93 are the two well-known sites of heme binding in water-dissolved holo-myoglobin, with His93 being a proximal, strongly binding partner, while the distal His64 weakly coordinates to the heme through a small-molecule ligand, e.g., water or O(2). The heme bonding scheme in a water-free environment is as yet unclear. Here we employed electron transfer dissociation tandem mass spectrometry to study the preferential attachment site of the ferri-heme (Fe(3+)) in electrospray-produced 12+, 14+, and 16+ holo-myoglobin ions. Contrary to expectations, in lower-charge complexes that should have a structure resembling that in solution, the heme seems to be preferentially attached to the "distal" histidine. In contrast, in the highest studied charge state, the "proximal" histidine is the site of preferential attachment; the 14+ charge state is an intermediate case. This surprising finding raises a question of heme coordination in proteins transferred to water-free environment, as well as the effect of the protonation sites on heme bonding.

New proteomic developments to analyze protein isomerization and their biological significance in plants.

Spontaneous isoaspartyl formation from aspartyl dehydration or asparaginyl deamidation is a major source of modifications in protein structures. In cells, these conformational changes could be reverted by the protein L-isoaspartyl methyltransferase (PIMT) repair enzyme that converts the isoaspartyl residues into aspartyl. The physiological importance of this metabolism has been recently illustrated in plants. Recent developments allowing peptide isomer identification and quantification at the proteome scale are portrayed. The relevance of these new proteomic approaches based on 2-D electrophoresis or electron capture dissociation analysis methods was initially documented in mammals. Extended use to Arabidopsis model systems is promising for the discovery of controlling mechanisms induced by these particular post-translational modifications and their biological role in plants.

Calibration function for the Orbitrap FTMS accounting for the space charge effect.

Ion storage in an electrostatic trap has been implemented with the introduction of the Orbitrap Fourier transform mass spectrometer (FTMS), which demonstrates performance similar to high-field ion cyclotron resonance MS. High mass spectral characteristics resulted in rapid acceptance of the Orbitrap FTMS for Life Sciences applications. The basics of Orbitrap operation are well documented; however, like in any ion trap MS technology, its performance is limited by interactions between the ion clouds. These interactions result in ion cloud couplings, systematic errors in measured masses, interference between ion clouds of different size yet with close m/z ratios, etc. In this work, we have characterized the space-charge effect on the measured frequency for the Orbitrap FTMS, looking for the possibility to achieve sub-ppm levels of mass measurement accuracy (MMA) for peptides in a wide range of total ion population. As a result of this characterization, we proposed an m/z calibration law for the Orbitrap FTMS that accounts for the total ion population present in the trap during a data acquisition event. Using this law, we were able to achieve a zero-space charge MMA limit of 80 ppb for the commercial Orbitrap FTMS system and sub-ppm level of MMA over a wide range of total ion populations with the automatic gain control values varying from 10 to 10(7).

Evolution of a domain conserved in microtubule-associated proteins of eukaryotes.

The microtubule network, the major organelle of the eukaryotic cytoskeleton, is involved in cell division and differentiation but also with many other cellular functions. In plants, microtubules seem to be involved in the ordered deposition of cellulose microfibrils by a so far unknown mechanism. Microtubule-associated proteins (MAP) typically contain various domains targeting or binding proteins with different functions to microtubules. Here we have investigated a proposed microtubule-targeting domain, TPX2, first identified in the Kinesin-like protein 2 in Xenopus. A TPX2 containing microtubule binding protein, PttMAP20, has been recently identified in poplar tissues undergoing xylogenesis. Furthermore, the herbicide 2,6-dichlorobenzonitrile (DCB), which is a known inhibitor of cellulose synthesis, was shown to bind specifically to PttMAP20. It is thus possible that PttMAP20 may have a role in coupling cellulose biosynthesis and the microtubular networks in poplar secondary cell walls. In order to get more insight into the occurrence, evolution and potential functions of TPX2-containing proteins we have carried out bioinformatic analysis for all genes so far found to encode TPX2 domains with special reference to poplar PttMAP20 and its putative orthologs in other plants.