The existence of multiple variants with differences in either charge, molecular weight or other properties is a common feature of monoclonal antibodies. These charge variants are generally referred to as acidic or basic compared with the main species. The chemical nature of the main species is usually well-understood, but understanding the chemical nature of acidic and basic species, and the differences between all three species, is critical for process development and formulation design. Complete understanding of acidic and basic species, however, is challenging because both species are known to contain multiple modifications, and it is likely that more modifications may be discovered. This review focuses on the current understanding of the modifications that can result in the generation of acidic and basic species and their affect on antibody structure, stability and biological functions. Chromatography elution profiles and several critical aspects regarding fraction collection and sample preparations necessary for detailed characterization are also discussed.

For improved detection of diverse posttranslational modifications (PTMs), direct fragmentation of protein ions by top down mass spectrometry holds promise but has yet to be achieved on a large scale. Using lysate from Saccharomyces cerevisiae, 117 gene products were identified with 100% sequence coverage revealing 26 acetylations, 1 N-terminal dimethylation, 1 phosphorylation, 18 duplicate genes, and 44 proteolytic fragments. The platform for this study combined continuous-elution gel electrophoresis, reversed-phase liquid chromatography, automated nanospray coupled with a quadrupole-FT hybrid mass spectrometer, and a new search engine for querying a custom database. The proteins identified required no manual validation, ranged from 5 to 39 kDa, had codon biases from 0.93 to 0.083, and were primarily associated with glycolysis and protein synthesis. Illustrations of gene-specific identifications, PTM detection and subsequent PTM localization (using either electron capture dissociation or known PTM data stored in a database) show how larger scale proteome projects incorporating top down may proceed in the future using commercial Q-FT instruments.

The extension of quantitation methods for small peptides to ions above 5 kDa, and eventually to global quantitative proteomics of intact proteins, will require extensive refinement of current analytical approaches. Here we evaluate postgrowth Cys-labeling and 14N/15N metabolic labeling strategies for determination of relative protein expression levels and their posttranslational modifications using top-down mass spectrometry (MS). We show that intact proteins that are differentially alkylated with acrylamide (+71 Da) versus iodoacetamide (+57 Da) have substantial chromatographic shifts during reversed-phase liquid chromatography separation (particularly in peak tails), indicating a requirement for stable isotopes in alkylation tags for top-down MS. In the 14N/15N metabolic labeling strategy, we achieve 98% 15N incorporation in yeast grown 10 generations under aerobic conditions and determine 50 expression ratios using Fourier transform ion cyclotron resonance MS in comparing these cells to anaerobically grown control (14N) cells. We devise quantitative methods for top-down analyses, including a correction factor for accurate protein ratio determination based upon the signal-to-noise ratio. Using a database of 200 yeast protein forms identified previously by top-down MS, we verify the intact mass tag concept for protein identification without tandem MS. Overall, we find that top-down MS promises work flows capable of large-scale proteome profiling using stable isotope labeling and the determination of >5 protein ratios per spectrum.

An automated top-down approach including data-dependent MS 3 experiment for protein identification/characterization is described. A mixture of wild-type yeast proteins has been separated on-line using reverse-phase liquid chromatography and introduced into a hybrid linear ion trap (LTQ) Fourier transform ion cylclotron resonance (FTICR) mass spectrometer, where the most abundant molecular ions were automatically isolated and fragmented. The MS 2 spectra were interpreted by an automated algorithm and the resulting fragment mass values were uploaded to the ProSight PTM search engine to identify three yeast proteins, two of which were found to be modified. Subsequent MS 3 analyses pinpointed the location of these modifications. In addition, data-dependent MS 3 experiments were performed on standard proteins and wild-type yeast proteins using the stand alone linear trap mass spectrometer. Initially, the most abundant molecular ions underwent collisionally activated dissociation, followed by data-dependent dissociation of only those MS 2 fragment ions for which a charge state could be automatically determined. The resulting spectra were processed to identify amino acid sequence tags in a robust fashion. New hybrid search modes utilized the MS 3 sequence tag and the absolute mass values of the MS 2 fragment ions to collectively provide unambiguous identification of the standard and wild-type yeast proteins from custom databases harboring a large number of post-translational modifications populated in a combinatorial fashion Protein identification via mass spectrometry (MS) mainly relies on two general strategies. With the bottom-up approach, proteins, purified or in complex mixtures, are proteolytically or chemically digested, followed by analysis using MS and tandem MS (MS/MS) of the resulting peptides, with identification provided by a database search of the product ion MS/ MS spectra [1,2]. Alternatively, with the top-down approach, the intact protein ions, individually or in mixtures, are mass analyzed and then fragmented inside the mass spectrometer without prior digestion [3,4]. The advantage of the latter method is the ability to measure the intact protein molecular weight, thus preserving both the protein sequence and the integrity of most post-translational modifications [5,6]. This allows one to proceed from protein identification to primary sequence characterization in the same experimental dataset.With a few exceptions [7][8][9][10] to date, the top-down approach has been restricted to FTICR instruments because of the need for high resolving power and mass accuracy for protein identification and characterization via accurate mass analysis of the intact protein molecular ions and their fragment ions. Intact protein and fragment molecular weights can be searched against a corresponding database in a manner similar to that of the bottom-up approach to provide protein identification [11][12][13]. At the moment, ProSight PTM is the only available database search engine for top-down MS [14,15]. The proba...

N-glycosylation of immunoglobulin G (IgG) at asparigine residue 297 plays a critical role in antibody stability and immune cell-mediated Fc effector function. Current understanding pertaining to Fc glycosylation is based on studies with IgGs that are either fully glycosylated [both heavy chain (HC) glycosylated] or aglycosylated (neither HC glycosylated). No study has been reported on the properties of hemi-glycosylated IgGs, antibodies with asymmetrical glycosylation in the Fc region such that one HC is glycosylated and the other is aglycosylated. We report here for the first time a detailed study of how hemi-glycosylation affects the stability and functional activities of an IgG1 antibody, mAb-X, in comparison to its fully glycosylated counterpart. Our results show that hemi-glycosylation does not impact Fab-mediated antigen binding, nor does it impact neonatal Fc receptor binding. Hemi-glycosylated mAb-X has slightly decreased thermal stability in the CH2 domain and a moderate decrease (∼20%) in C1q binding. More importantly, the hemi-glycosylated form shows significantly decreased binding affinities toward all Fc gamma receptors (FcγRs) including the high-affinity FcγRI, and the low-affinity FcγRIIA, FcγRIIB, FcγRIIIA and FcγRIIIB. The decreased binding affinities to FcγRs result in a 3.5-fold decrease in antibody-dependent cell cytotoxicity (ADCC). As ADCC often plays an important role in therapeutic antibody efficacy, glycosylation status will not only affect the antibody quality but also may impact the biological function of the product.

Disease modifying treatments for Alzheimer’s disease (AD) constitute a major goal in medicine. Current trends suggest that biomarkers reflective of AD neuropathology and modifiable by treatment would provide supportive evidence for disease modification. Nevertheless, a lack of quantitative tools to assess disease modifying treatment effects remains a major hurdle. Cerebrospinal fluid (CSF) biochemical markers such as total tau, p-tau and Ab42 are well established markers of AD; however, global quantitative biochemical changes in CSF in AD disease progression remain largely uncharacterized. Here we applied a high resolution open discovery platform, dMS, to profile a cross-sectional cohort of lumbar CSF from post-mortem diagnosed AD patients versus those from non-AD/non-demented (control) patients. Multiple markers were identified to be statistically significant in the cohort tested. We selected two markers SME-1 (p<0.0001) and SME-2 (p = 0.0004) for evaluation in a second independent longitudinal cohort of human CSF from post-mortem diagnosed AD patients and age-matched and case-matched control patients. In cohort-2, SME-1, identified as neuronal secretory protein VGF, and SME-2, identified as neuronal pentraxin receptor-1 (NPTXR), in AD were 21% (p = 0.039) and 17% (p = 0.026) lower, at baseline, respectively, than in controls. Linear mixed model analysis in the longitudinal cohort estimate a decrease in the levels of VGF and NPTXR at the rate of 10.9% and 6.9% per year in the AD patients, whereas both markers increased in controls. Because these markers are detected by mass spectrometry without the need for antibody reagents, targeted MS based assays provide a clear translation path for evaluating selected AD disease-progression markers with high analytical precision in the clinic.

Complete coverage of protein primary structure is demonstrated for 37 yeast protein forms between 6 and 30 kDa in an improved platform for Top Down mass spectrometry (MS). Tandem mass spectrometry (MS/MS) for protein identification with 100% sequence coverage is achieved in a highly automated fashion with 15-300-fold less sample amounts than an initial report of a proteome fractionation approach employing preparative gel electrophoresis with an acid-labile surfactant to facilitate reversed phase separation in a second dimension. Using a quadrupole-enhanced Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FTICRMS) improves the dynamic range for protein detection by ~50-fold and MS/MS by ~30-fold. The technology development illustrated here typifies an accelerating effort to detect whole proteins in a more general and higher throughput fashion for improved biomarker identification and detection of diverse post-translational modifications. Capillary RPLC is used in both off-line and on-line modes, with one on-line LC/FTMS sample providing 25 observed protein forms from 11 to 22 kDa.

Dear Editor, Methyl-CpG-binding protein 2 (MeCP2) is a ubiquitously expressed nuclear protein originally identified as a methylated DNA binding protein, which is particularly abundant in mature neurons 1,2. Deficiency or excess of MeCP2 causes severe neurological problems. Mutations in MeCP2 account for 95% of the dominant X-linked neurological disorder Rett syndrome 3. MeCP2 have two key functional domains: the methyl-DNA binding domain (MBD) and the transcriptional repressor domain (TRD). Almost all of missense Rett mutations are clustered in these two domains, such as R133C, F155S, T158M in MBD, and R306H in TRD 4,5. The mechanism of the mutations leading to Rett syndrome is still not well understood. Here, we reveal that MeCP2 can drive the liquid-liquid phase separation (LLPS) in complex with DNA. Interestingly, this ability is compromised in the presence of mutations found in Rett syndrome patients, suggesting a potential common mechanism by disrupting LLPS of MeCP2 droplets underlying Rett syndrome. MeCP2 forms sharp condensed foci which highly overlap with DNA dense compartments in neuronal nuclei 6,7. Recently, LLPS has been recognized as an important mechanism to condensate molecules to form membraneless compartments within a cell 8. As both the MBD and TRD of MeCP2 bind to DNA 9 , we hypothesized that the sharp puncta of MeCP2 in the nuclei were phase separated liquid droplets mediated by multivalent interactions between MeCP2 and DNA. To test this hypothesis, we purified full-length recombinant His-MBP-MeCP2 (mouse MeCP2-e2 if not specified) and released