Hongqian Yang

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.

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.

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.

Blood Plasma IgG Fc Glycans are Significantly Altered in Alzheimer's Disease and Progressive Mild Cognitive Impairment

Blood-based anti-amyloid-β (Aβ) immunoglobulins (IgGs) and peripheral inflammation are factors correlating with development of Alzheimers 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.

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 107.

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 O2. 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 (Fe3+) 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.

Prognostic Polypeptide Blood Plasma Biomarkers of Alzheimer's Disease Progression

BACKGROUND Patients with mild cognitive impairment (MCI) have varying risks of progression to Alzheimers 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.

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 Alzheimers 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.

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

BackgroundFocal 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.MethodsUrinary 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.ResultsTwenty 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.ConclusionThe 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.

Multiple Soft Ionization of Gas‐Phase Proteins and Swift Backbone Dissociation in Collisions with ≤99 eV Electrons

Mass spectrometry (MS) is based on the ionization of sample molecules and subsequent measurement of their mass-tocharge ratio m/z. For measurements of molecular mass, it is vital to preserve the intact molecular ion after ionization, that is, to perform “soft ionization”. Since the end of the 1980s, large (> 10 kDa) biomolecules have been subjected to soft ionization by either electrospray ionization (ESI) or matrixassisted laser desorption ionization (MALDI), which both add protons to neutral molecules in the positive-ion mode. With the emergence of ESI and MALDI, the challenge has shifted to informative fragmentation of polypeptides. In particular, “native mass spectrometry” produces intact protein–protein and protein–molecule complexes in low protonation states; such complexes fragment poorly and thus yield little structural information. Facile fragmentation of such species requires the rapid introduction of additional charges or radical sites, or preferably both of these types of bond weakening simultaneously. Before the emergence of soft-ionization techniques, gasphase cations of biomolecules were predominantly obtained by electron ionization (EI) in which 70 eV electrons remove one valence electron from gas-phase neutral molecules. Whereas small molecules can be ionized by electrons two or three times, 9] for molecules larger than 1 kDa, attempts to obtain even singly charged molecular ions failed initially. However, in the 1990s it was shown that protonated biopolymers can increase their charge state by one unit in gas-phase collisions with electrons that have an energy higher than 11 eV. Recently, our research group demonstrated that above 40 eV, double ionization can occur for molecules as large as ubiquitin (8.6 kDa). However, for small proteins, the yield in EI of intact molecular species was relatively small in comparison to the yield of species derived from the dominant EI-induced dissociation. Herein, we report that for larger (> 10 kDa) protein ions, multiple ionization can occur without the fragmentation of molecular species. Moreover, protein–molecule complexes, such as holomyoglobin, retain elements of their tertiary structure even after four ionizations, and when backbone fragmentation occurs, polypeptide fragments can preserve weak interactions with the noncovalently bound molecule. Figure 1 presents the EI mass spectrum of cytochrome c, a 12.4 kDa single polypeptide chain with a covalently attached heme group. Upon irradiation with approximately 50 eV