Light contamination in stable isotope-labelled internal peptide standards is frequent and a potential source of false discovery and quantitation error in proteomics

In mass spectrometry-based proteomics, heavy internal standards are used to validate target peptide detections and to calibrate peptide quantification. Here we report light contamination present in heavy labelled synthetic peptides of high isotopic enrichment. Application of such peptides as assay-internal standards potentially compromises the detection and quantification especially of low abundant cellular peptides. Therefore, it is important to adopt guidelines to prevent false-positive identifications of endogenous light peptides as well as errors in their quantification from biological samples.

One-step Isolation of Protein C-terminal Peptides from V8 Protease-digested Proteins by Metal Oxide-based Ligand Exchange Chromatography

We have developed a one-step method to isolate protein C-terminal peptides from V8 protease-digested proteins by metal oxide-based ligand-exchange (MOLEX) chromatography. V8 protease cleaves the C-terminal side of Asp and Glu, affording a digested peptide with two carboxy groups at the C-terminus, whereas the protein C-terminal peptide has only one α-carboxy group. In MOLEX chromatography, a stable chelate is formed between dicarboxylates and metal atoms, so that the non-terminal (i.e., internal) peptide is retained, whereas the protein C-terminal peptide flows through the MOLEX column. After optimization of the MOLEX chromatographic conditions, 1619 protein C-termini were identified from 30 μg of peptides (10 μg each, in triplicate) derived from human HeLa cells by means of nanoLC/MS/MS. When the MOLEX-isolated sample from 200 μg of HeLa peptides was further divided into six fractions by high-pH reversed-phase LC prior to nanoLC/MS/MS, 2202 protein C-termini were identified with less than 3% contamination with internal peptides. We believe this is the largest coverage with the highest purity reported to date in human protein C-terminomics. This fast, simple, sensitive and selective method to isolate protein C-terminal peptides should be useful for profiling protein C-termini on a proteome-wide scale.

Bio-inspired gene carriers with low cytotoxicity constructed via the assembly of dextran nanogels and nano-coacervates

Aim: To achieve safe and biocompatible gene carriers. Materials & methods: A core/shell-structured hierarchical carrier with an internal peptide/gene coacervate ‘core’ and a dextran nanogel ‘shell’ on the surface has been designed. Results: The dextran nanogels shield coacervate (DNSC) can effectively condense genes and release them in reducing environments. The dextran nanogel-based ‘shell’ can effectively shield the positive charge of the peptide/gene coacervate ‘core’, thus reducing the side effects of cationic gene carriers. In contrast with the common nonviral gene carriers that had high cytotoxicities, the DNSC showed a high transfection efficiency while maintaining a low cytotoxicity. Conclusion: The DNSC provides an effective environmentally responsive gene carrier with potential applications in the fields of gene therapy and gene carrier development.

Adjunctive Immunotherapeutic Efficacy of N-Formylated Internal Peptide of Mycobacterial Glutamine Synthetase in Mouse Model of Tuberculosis

Background: Host-directed therapies are a comparatively new and promising method for the treatment of tuberculosis. A variety of host pathways, vaccines and drugs have the potential to provide novel adjunctive therapies for the treatment of tuberculosis. In this connection, we have earlier reported the immunotherapeutic potential of N-formylated N-terminal peptide of glutamine synthetase of Mycobacterim tuberculosis H37Rv (Mir SA and Sharma S, 2014). Now in the present study, we investigated the immunotherapeutic effect of N-terminally formylated internal-peptide 'f- MLLLPD' of mycobacterial glutamine synthetase (Rv2220) in mouse model of tuberculosis. Methods: The N-terminally formylated peptide, f-MLLLPD was tested for its potential to generate Reactive Oxygen Species (ROS) in murine neutrophils. Further, its therapeutic effect alone or in combination with anti-tubercular drugs was evaluated in mouse model of tuberculosis. Results: The f-MLLLPD peptide treatment alone and in combination with ATDs reduced the bacterial load (indicated as colony forming units) in lungs of infected mice by 0.58 (p<0.01) and 2.92 (p<0.001) log10 units respectively and in their spleens by 0.46 (p<0.05) and 2.46 (p<0.001) log10 units respectively. In addition, the observed histopathological results correlated well with the CFU data. Conclusion : The results of the current study show that f-MLLLPD peptide confers an additional therapeutic efficacy to the anti-tuberculosis drugs.

Development of Targeted Mass Spectrometry-Based Approaches for Quantitation of Proteins Enriched in the Postsynaptic Density (PSD)

The postsynaptic density (PSD) is a structural, electron-dense region of excitatory glutamatergic synapses, which is involved in a variety of cellular and signaling processes in neurons. The PSD is comprised of a large network of proteins, many of which have been implicated in a wide variety of neuropsychiatric disorders. Biochemical fractionation combined with mass spectrometry analyses have enabled an in-depth understanding of the protein composition of the PSD. However, the PSD composition may change rapidly in response to stimuli, and robust and reproducible methods to thoroughly quantify changes in protein abundance are warranted. Here, we report on the development of two types of targeted mass spectrometry-based assays for quantitation of PSD-enriched proteins. In total, we quantified 50 PSD proteins in a targeted, parallel reaction monitoring (PRM) assay using heavy-labeled, synthetic internal peptide standards and identified and quantified over 2100 proteins through a pre-determined spectral library using a data-independent acquisition (DIA) approach in PSD fractions isolated from mouse cortical brain tissue.

Structural basis of internal peptide recognition by PDZ domains using molecular dynamics simulations

PDZ domains are important peptide recognition modules which usually recognize short C-terminal stretches of their interaction partners, but certain PDZ domains can also recognize internal peptides in the interacting proteins. Due to the scarcity of data on internal peptide recognition and lack of understanding of the mechanistic details of internal peptide recognition, identification of PDZ domains capable of recognizing internal peptides has been a difficult task. Since Par-6 PDZ domain can recognize both C-terminal and internal peptides, we have carried out multiple explicit solvent MD simulations of 1 μs duration on free and peptide bound Par-6 PDZ to decipher mechanistic details of internal peptide recognition. These simulations have been analyzed to identify residues which play a crucial role in internal peptide recognition by PDZ domains. Based on the conservation profile of the identified residues, we have predicted 47 human PDZ domains to be capable of recognizing internal peptides in human. We have also investigated how binding of CDC42 to the CRIB domain adjacent to the Par6 PDZ allosterically modulate the peptide recognition by Par6 PDZ. Our MD simulations on CRIB-Par6_PDZ di-domain in isolation as well as in complex with CDC42, indicate that in absence of CDC42 the adjacent CRIB domain induces open loop conformation of PDZ facilitating internal peptide recognition. On the other hand, upon binding of CDC42 to the CRIB domain, Par6 PDZ adopts closed loop conformation required for recognition of C-terminus peptides. These results provide atomistic details of how binding of interaction partners onto adjacent domains can allosterically regulate substrate binding to PDZ domains. In summary, MD simulations provide novel insights into the modulation of substrate recognition preference of PDZ by specific peptides, adjacent domains and binding of interaction partners at allosteric sites.

Quantification of Serum IgG Subclasses by Use of Subclass-Specific Tryptic Peptides and Liquid Chromatography–Tandem Mass Spectrometry

BACKGROUND Measurement of IgG subclasses is a useful tool for investigation of humoral immune deficiency in the presence of total IgG within reference intervals and IgG4-related disease. Nephelometry has been the method of choice for quantification. We describe an LC-MS/MS method that can multiplex all 4 subclasses along with total IgG by use of either IgG subclass-specific peptide stable isotope–labeled internal standards or a surrogate digest standard for quantification and does not rely on antigen/antibody reactions. METHODS We combined serum with labeled internal peptide standards and intact purified horse IgG. Samples were denatured, reduced, alkylated, and digested. We analyzed the digested serum by LC-MS/MS for IgG subclasses 1–4 and total IgG. RESULTS We assayed 112 patient sera by LC-MS/MS and immunonephelometry. The mean of the slopes and R2 values for IgG1, IgG2, IgG3, IgG4, and IgG were 1.18 and 0.93, respectively. Interassay imprecision for the LC-MS/MS method was &lt;15% for total IgG and subclasses and was slightly improved by use of a calibrator peptide from an exogenous horse IgG. Summed total IgG correlated with the measured total IgG within 10%. Reference intervals and analytical measuring range were all similar to our previous validation data for the immunonephelometry assays. CONCLUSIONS Total IgG and IgG subclasses 1, 2, 3, and 4 can be quantified by LC-MS/MS with performance comparable to nephelometry.