Mass Spectrometric Analysis Of Proteins Research Articles

Development and validation of an ELISA to measure regenerating island-derived protein 3E in canine blood.

Regenerating island-derived proteins (REG) are upregulated in people with sepsis, pancreatitis, and gastrointestinal diseases. One member of the REG family, namely REG3E, was recently identified in pancreatic tissue and plasma of dogs, with high expression in pancreatitis and sepsis. We aimed to develop and validate an ELISA to measure REG3E concentrations in canine blood. An indirect sandwich ELISA was developed using recombinant canine REG3E protein and polyclonal anti-canine REG3E antibodies raised in guinea pigs and rabbits. Antibody specificity was assessed using western blot and mass spectrometric analysis of protein purified from canine plasma. Assay validation included evaluation of dilutional linearity, parallelism, spiking recovery, repeatability and reproducibility, stability, interferences, and comparison of serum and heparinized plasma. Antibodies bound specifically to REG3E with no evidence of cross-reactivity with other proteins. The limit of detection of the ELISA was 15 ng/mL, and the lower limit of quantification was 30 ng/mL. The assay demonstrated good to excellent linearity, dilutional and mixing parallelism, and recovery, with mean observed-to-expected ratios of 104%, 107%, 102%, and 92%, respectively, and no evidence of a hook effect. Coefficients of variation were ≤8.5% for repeatability and ≤14.3% for reproducibility at three different levels. Measurements of REG3E in plasma were not significantly influenced by different storage conditions, freeze-thawing cycles, hemolysis, lipemia, or icterus. There was no significant difference between REG3E concentrations in heparinized plasma and serum samples. The canine REG3E ELISA has acceptable precision, accuracy, linearity, and reproducibility for the measurement of REG3E in canine plasma and serum.

A 3D-Printed Offline Nano-ESI Source for Bruker MS Instruments.

Nanoelectrospray ionization (nano-ESI) is a highly efficient and a widely used technique for the ionization of minute amounts of analyte. Offline nano-ESI sources are convenient for the direct infusion of complex mixtures that suffer from high matrix content and are crucial for the native mass spectrometric analysis of proteins. For Bruker instruments, no such source is readily available. Here we close this gap and present a 3D-printable nano-ESI source for Bruker instruments, which can be assembled by anyone with access to 3D printers. The source can be fitted to any Bruker mass spectrometer with an ionBooster ESI source and only requires minor, reversible changes to the original Bruker hardware. The general utility was demonstrated by recording high-resolution MS spectra of small molecules, intact proteins, as well as complex biological samples in negative and positive ion mode on two different Bruker instruments.

A Fluid Multivalent Magnetic Interface for High-Performance Isolation and Proteomic Profiling of Tumor-Derived Extracellular Vesicles.

Isolation and analysis of tumor-derived extracellular vesicles (T-EVs) are important for clinical cancer management. Here, we develop a fluid multivalent magnetic interface (FluidmagFace) in a microfluidic chip for high-performance isolation, release, and protein profiling of T-EVs. The FluidmagFace increases affinity by 105-fold with fluidity-enhanced multivalent binding to improve isolation efficiency by 13.9% compared with a non-fluid interface. Its anti-adsorption property and microfluidic hydrodynamic shear minimize contamination, increasing detection sensitivity by two orders of magnitude. Moreover, its reversibility and expandability allow high-throughput recovery of T-EVs for mass spectrometric protein analysis. With the chip, T-EVs are detected in all tested cancer samples with identification of differentially expressed proteins compared with healthy controls. The FluidmagFace opens a new avenue to isolation and release of targets for cancer diagnosis and biomarker discovery.

A Fluid Multivalent Magnetic Interface for High‐Performance Isolation and Proteomic Profiling of Tumor‐Derived Extracellular Vesicles

AbstractIsolation and analysis of tumor‐derived extracellular vesicles (T‐EVs) are important for clinical cancer management. Here, we develop a fluid multivalent magnetic interface (FluidmagFace) in a microfluidic chip for high‐performance isolation, release, and protein profiling of T‐EVs. The FluidmagFace increases affinity by 105‐fold with fluidity‐enhanced multivalent binding to improve isolation efficiency by 13.9 % compared with a non‐fluid interface. Its anti‐adsorption property and microfluidic hydrodynamic shear minimize contamination, increasing detection sensitivity by two orders of magnitude. Moreover, its reversibility and expandability allow high‐throughput recovery of T‐EVs for mass spectrometric protein analysis. With the chip, T‐EVs were detected in all tested cancer samples with identification of differentially expressed proteins compared with healthy controls. The FluidmagFace opens a new avenue to isolation and release of targets for cancer diagnosis and biomarker discovery.

Tnpo3 enables EBF1 function in conditions of antagonistic Notch signaling.

Transcription factor EBF1 (early B cell factor 1) acts as a key regulator of B cell specification. The transcriptional network in which EBF1 operates has been extensively studied; however, the regulation of EBF1 function remains poorly defined. By mass spectrometric analysis of proteins associated with endogenous EBF1 in pro-B cells, we identified the nuclear import receptor Transportin-3 (Tnpo3) and found that it interacts with the immunoglobulin-like fold domain of EBF1. We delineated glutamic acid 271 of EBF1 as a critical residue for the association with Tnpo3. EBF1E271A showed normal nuclear localization; however, it had an impaired B cell programming ability in conditions of Notch signaling, as determined by retroviral transduction of Ebf1 -/- progenitors. By RNA-seq analysis of EBF1E271A-expressing progenitors, we found an up-regulation of T lineage determinants and down-regulation of early B genes, although similar chromatin binding of EBF1E271A and EBF1wt was detected in pro-B cells expressing activated Notch1. B lineage-specific inactivation of Tnpo3 in mice resulted in a block of early B cell differentiation, accompanied by a down-regulation of B lineage genes and up-regulation of T and NK lineage genes. Taken together, our observations suggest that Tnpo3 ensures B cell programming by EBF1 in nonpermissive conditions.

Comparative Proteomics of Lipid Transport Proteins and Assessment of Oxidative Stress Parameters in Acute Ischemic Stroke and Healthy Control

Background: Stroke is one of the major causes of mortality worldwide. Ischemic stroke occurs due to blood vessel blockage that limits blood supply to the brain. The present study aims to identify the serum proteins in terms of its lipid transportation function and the level of oxidative stress parameters. The study was done in human blood samples of ischemic stroke patients and healthy control participants. The sample size used was 26 in each group for oxidative stress study and 3 per group for mass spectrometric protein analysis. The study was designed as a comparative analysis of oxidative stress parameters between control and ischemic stroke. The in gel tryptic digestion followed by Liquid chromatography – mass spectrometry (LC-MS-MS) analysis was done to identify the differentially expressed serum proteins between control and stroke. Students t test was used for comparison between groups and p value less than 0.05 (p<.05) considered as significant. The study observed that the antioxidant parameters such as catalase and glutathione had lowered activity in ischemic stroke group compared to healthy control and malondialdehyde levels were higher in ischemic stroke compared to control. From the in gel tryptic digestion, we observed that proteins linked to lipid transport and inflammation had differential expression between stroke and control. The serum proteins which were differentially altered in 25 KDa region in electrophoretogram between control and stroke had functional association with lipid transportation process and stress associated inflammatory roles.

Proteomic analysis of host cellular proteins co-immunoprecipitated with duck enteritis virus gC

Duck enteritis virus (DEV), the causative agent of duck viral enteritis, causes a contagious, lethal viral disease in Anseriformes (waterfowls). In virus infection, host-virus interaction plays a crucial role in virus replication and pathogenesis. In our previous study, mRFP was fused with the C-terminus of DEV glycoprotein C (gC) to construct a fluorescent-tag DEV virus rgCRFP. In the current study, fluorescent fusion protein (gC-mRFP) was used as the proteomic probe. Co-immunoprecipitation and mass spectrometric analysis of proteins from rgCRFP-infected chicken embryo fibroblasts using commercial anti-RFP antibody led to the identification of a total of 21 gC interacting host proteins. Out of these 21 proteins, the interaction of seven host proteins (GNG2, AR1H1, PPP2CA, UBE2I, MCM5, NUBP1, HN1) with DEV gC protein was validated using membrane-bound split-ubiquitin yeast two-hybrid system (MbYTH) and bimolecular fluorescence complementation (BiFC) analyses. It indicated direct interaction between these proteins with DEV gC protein. This study has furthered the current understanding of DEV virus infection and pathogenesis. SignificancegC is an crucial glycoprotein of duck enteritis virus that plays an important role in the viral life cycle. Uncovering the interaction between virus-host is very important to elucidate the pathogenic mechanism of the virus. In this study, host factors interacting with DEV gC have been discerned. And seven host proteins (GNG2, AR1H1, PPP2CA, UBE2I, MCM5, NUBP1, HN1) have been further validated to interact with DEV gC using MbYTH and BiFC analyses. These outcomes could shed light on how DEV manipulates the cellular machinery, which could further our understanding of DEV pathogenesis.

Mass-Spectrometric MRM Analysis of FDA-Approved Proteins in Plasma of Healthy Volunteers

The proteomic composition of a biological sample serves as the most important feature of a biological object, and it allows discriminating normal and pathological conditions. Targeted mass spectrometric analysis, particularly, multiple reaction monitoring (MRM) using synthetic stable isotope-labeled internal standards (SIS), is the main alternative to the ELISA method for analysis of diagnostically significant proteins. Based on the MRM results, a prototype test system has been developed; it employs the targeted mass spectrometric method for multiplex, quantitative analysis of FDA-approved proteins in whole plasma. Using this approach, it was possible to measure the content of 42 proteins in 31 samples in a concentration range spanning five orders of magnitude. The interindividual variability for 30 of the 42 registered proteins was less than 40%. The largest scatter was observed for haptoglobin (68%), immunoglobulin heavy constant delta IGHD (90%), angiotensin (72%), sex hormone-binding globulin SHBG (100%), and lipoprotein-(a) (136%). The obtained results on the concentration of proteins correlate with published data (Hortin et al., Clinical Chemistry, 2008, vol. 54, 1608) with R2 = 0.84. The developed prototype test system based on targeted mass spectrometric analysis of proteins can be considered as an alternative to methods using monoclonal antibodies.

Differential cleavage of viral polypeptides by allotypic variants of granzyme B skews immunity to mouse cytomegalovirus

We investigated the molecular basis for the remarkably different survival outcomes of mice expressing different alloforms of the pro-apoptotic serine protease granzyme B to mouse cytomegalovirus infection. Whereas C57BL/6 mice homozygous for granzyme BP (GzmBP/P) raise cytotoxic T lymphocytes that efficiently kill infected cells, those of C57BL/6 mice congenic for the outbred allele (GzmBW/W) fail to kill MCMV-infected cells and died from uncontrolled hepatocyte infection and acute liver failure. We identified subtle differences in how GzmBP and GzmBW activate cell death signalling - both alloforms predominantly activated pro-caspases directly, and cleaved pro-apoptotic Bid poorly. Consequently, neither alloform initiated mitochondrial outer membrane permeabilization, or was blocked by Bcl-2, Bcl-XL or co-expression of MCMV proteins M38.5/M41.1, which together stabilize mitochondria by sequestering Bak/Bax. Remarkably, mass spectrometric analysis of proteins from MCMV-infected primary mouse embryonic fibroblasts identified 13 cleavage sites in nine viral proteins (M18, M25, M28, M45, M80, M98, M102, M155, M164) that were cleaved >20-fold more efficiently by either GzmBP or GzmBW. Notably, M18, M28, M45, M80, M98, M102 and M164 were cleaved 20- >100-fold more efficiently by GzmBW, and so, would persist in infected cells targeted by CTLs from GzmBP/P mice. Conversely, M155 was cleaved >100-fold more efficiently by GzmBP, and would persist in cells targeted by CTLs of GzmBW/W mice. M25 was cleaved efficiently by both proteases, but at different sites. We conclude that different susceptibility to MCMV does not result from skewed endogenous cell death pathways, but rather, to as yet uncharacterised MCMV-intrinsic pathways that ultimately inhibit granzyme B-induced cell death.

Linker histone variant H1t is closely associated with repressed repeat-element chromatin domains in pachytene spermatocytes

BackgroundH1t is the major linker histone variant in pachytene spermatocytes, where it constitutes 50–60% of total H1. This linker histone variant was previously reported to localize in the nucleolar rDNA element in mouse spermatocytes. Our main aim was to determine the extra-nucleolar localization of this linker histone variant in pachytene spermatocytes.ResultsWe generated H1t-specific antibodies in rabbits and validated its specificity by multiple assays like ELISA, western blot, etc. Genome-wide occupancy studies, as determined by ChIP-sequencing in P20 mouse testicular cells revealed that H1t did not closely associate with active gene promoters and open chromatin regions. Annotation of H1t-bound genomic regions revealed that H1t is depleted from DSB hotspots and TSS, but are predominantly associated with retrotransposable repeat elements like LINE and LTR in pachytene spermatocytes. These chromatin domains are repressed based on co-association of H1t observed with methylated CpGs and repressive histone marks like H3K9me3 and H4K20me3 in vivo. Mass spectrometric analysis of proteins associated with H1t-containing oligonucleosomes identified piRNA–PIWI pathway proteins, repeat repression-associated proteins and heterochromatin proteins confirming the association with repressed repeat-element genomic regions. We validated the interaction of key proteins with H1t-containing oligonucleosomes by use of ChIP-western blot assays. On the other hand, we observe majority of H1t peaks to be associated with the intergenic spacer of the rDNA element, also in association with SINE elements of the rDNA element. Thus, we have identified the genomic and chromatin features of both nucleolar and extranucleolar localization patterns of linker histone H1t in the context of pachytene spermatocytes.ConclusionsH1t-containing repeat-element LINE and LTR chromatin domains are associated with repressive marks like methylated CpGs, histone modifications H3K9me3 and H4K20me3, and heterochromatin proteins like HP1β, Trim28, PIWIL1, etc. Apart from localization of H1t at the rDNA element, we demonstrate the extranucleolar association of this linker histone variant at repeat-associated chromatin domains in pachytene spermatocytes. We hypothesize that H1t might induce local chromatin relaxation to recruit heterochromatin and repeat repression-associated protein factors necessary for TE (transposable element) repression, the final biological effect being formation of closed chromatin repressed structures.

Mass-spectrometric MRM analysis of FDA-verified proteins in the blood plasma of healthy volunteers

The proteomic composition of a biological sample serves as the most important feature of a biological object, and it allows discriminating normal and pathological conditions. Targeted mass spectrometric analysis, namely, multiple reaction monitoring (MRM) using synthetic isotopically-labeled internal standard (SIS), is the main alternative to the ELISA method for the analysis of diagnostically significant proteins. Based on the MRM results, a prototype test system has been developed; it employs the targeted mass spectrometric method for multiplex, quantitative analysis of FDA-verified proteins in whole blood plasma. Using this approach, it was possible to measure the content of 42 proteins in 31 samples in a concentration range spanning five orders of magnitude. The interindividual variability for 30 of the 42 registered proteins was less than 40%. The largest scatter was observed for haptoglobin (68%), immunoglobulin heavy constant delta IGHD (90%), angiotensin (72%), sex hormone-binding globulin SHBG (100%) and lipoprotein-(a) (136%). The obtained results on the concentration of proteins correlate with published data (Hortin et al., 2008, Clinical Chemistry, 54, 1608) with R2=0.84. The developed prototype test system based on targeted mass spectrometric analysis of proteins can be considered as an alternative to methods using monoclonal antibodies.

Preservation of Protein Zwitterionic States in the Transition from Solution to Gas Phase Revealed by Sodium Adduction Mass Spectrometry.

The structural characterization of proteins and their interaction network mapping in the gas phase highlights the need to preserve their most nativelike conformers in the transition from the solution to gas phase. Zwitterionic interactions in a protein are weak bonds between oppositely charged residues, which make an important contribution to protein stability. However, it is still not clear whether the native zwitterionic states of proteins can be retained or not when it is transferred from the solution to gas phase. Using the nonspecific Na+ adduction as a novel signature, here we show that the zwitterionic states of proteins can be preserved when a moderated droplet desolvation condition (temperature <30 °C) is used in native electrospray ionization mass spectrometry. The very low-level nonspecific metal adduction to proteins under such conditions also enables rapid and direct determination of the binding states of metal-binding proteins and sensitive detection of proteins from solutions containing highly concentrated involatile salts (e.g., 50 mM NaCl). We believe that our findings can be instructive for performing mass spectrometric analysis of proteins and useful for protein ions desalting which simply involves altering the temperature and flow rate of drying gas in the desolvation region.

Abstract 5660: Proteomic comparative assessment of flash-frozen and OCT embedded breast cancer tissues for utilization in precision oncology discovery studies

Abstract Proteomic analysis is an important tool in classifying molecular architecture of tissue samples for subtyping and precision oncology applications. Tissues are commonly either flash frozen (FF) or embedded in optimal cutting temperature (OCT) compound for long-term storage. OCT provides a convenient specimen matrix for cryostat sectioning but contains glycols and resins which are incompatible with mass spectrometric analysis of proteins. In contrast, flash frozen preservation of tissue is historically considered to be more suitable and compatible with mass spectrometry applications. As a pilot study, we evaluated different tissue preservation methods, sample processing methods, and protein extraction methods, and their impact on quantitation and coverage of the proteome. In this pilot study, 30 breast cancer cases with matching flash frozen and OCT-embedded samples were used for proteomic analysis. Tissues were procured from the Clinical Breast Care Project (CBCP) in partnership with the Chan Soon-Shiong Institute of Molecular Medicine at Windber, and the Walter Reed National Military Medical Center (WRNMMC). Breast cancer subtypes were determined by clinical and expanded immunohistochemical panels. These subtypes were Her2, Luminal A (LA), Luminal B (LB) and Triple Negative (TN). Flash frozen tissues were cryosectioned to isolate 60-100 mg total tissue per case, and proteins were extracted using an 8M urea lysis buffer. The OCT specimens were laser microdissected to isolate primarily tumor cells from the samples. Proteins were extracted using the illustra TriplePrep kit. Digested proteins were then multiplexed using Tandem-Mass-Tag (TMT) 10plex isobaric labeling reagents, and chromatographically resolved using a Waters 2D nanoAcquity liquid chromatographer (LC) coupled to a Thermo Q Exactive Plus mass spectrometer (MS). Data was then analyzed using Proteome Discoverer. In total, 6130 proteins were identified using a 1% peptide FDR confidence filter in the 30 samples analyzed. Unsupervised hierarchical clustering of proteins across the samples resulted in two primary clusters, OCT and FF. Within the primary clustering, there were sub-clusters of Luminal, Her2, and Triple Negatives. At 2-fold threshold, 246 proteins were differentially expressed between OCT and FF tissues. However, the differential proteins between ER+ (LA and LB) and ER- (Her2 and TN) tumors correlate irrespective of the cryopreservation and processing methods. In conclusion, proteome analysis of samples stored under two different conditions provide subtyping information; however, there is differential expression of proteins between the two types of sample handling techniques which can impact biological interpretation of breast cancer precision oncology studies. Citation Format: Kiki Panagopoulos, Punit Shah, Albert Kovatich, Michael A. Kiebish, Jeffrey Hooke, Leigh Campbell, Mary Lou Cutler, Audrey Kovatich, Hai Hu, Brenda Deyarmin, Lori Sturtz, Praveen Kumar Raj Kumar, Stella Somiari, Emily Y. Chen, Elder Granger, Viatcheslav Akmaev, Rangaprasad Sarangarajan, Niven Narain, Craig D. Shriver. Proteomic comparative assessment of flash-frozen and OCT embedded breast cancer tissues for utilization in precision oncology discovery studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5660.

Mass Spectrometric Analysis of Proteins of L6 Skeletal Muscle Cells Under Different Glucose Conditions and Vitamin D Supplementation.

Type 2 Diabetes (T2D) is a metabolic disease which affects glucose homeostasis caused due to inability of the target cells to respond to insulin. Role of vitamin D in the pathogenesis and prevention of T2D has sparked widespread interest. Vitamin D plays a classical role in Ca++ homeostasis as well as regulates insulin secretion from β-cells and its action on various target cells. Proteins are the vital components of all cellular processes and their expression alters in response to various external or internal stimuli. Alteration in protein structure, function may contribute to the pathogenesis of many diseases including diabetes. Protein expression during the exposure of the cells to different glucose concentrations may alter and can give vital information about the pathogenesis of T2D. To study the effect of different glucose concentrations and supplementation of vitamin D on proteomic profile of L6 cell lines. L6 skeletal muscle cells were exposed to different Glucose (G) concentrations (0mM, 8mM, 16mM and 25mM) supplemented with Vitamin D (VD) for 48 hours. Total cell protein was extracted and protein profile was studied using SDS-PAGE. Three distinct bands observed in SDSPAGE in samples obtained from cells which were exposed to 8mM (G), 8mM (G) + VD and 16mM (G). The distinct bands were excised, in gel digestion were performed and MALDI-TOF analysis of the samples were done. MALDI-TOF analysis revealed these bands as mitochondrial uncoupling protein 3 (UCP3 MOUSE), Insulin gene enhancer protein 2 (ISL2 MOUSE) and Tubulin polyglutamylase complex1 (TPGS1 MOUSE) respectively. UCP3 protein is primarily expressed in the skeletal muscle cells and is involved in energy homeostasis and modulates insulin sensitivity. ISL2 protein plays an important role in differentiation and maintenance of the tissues. TPGS1 helps in microtubule polymerization might be helping in glucose transport and also play crucial role in the cellular movement, organization of intracellular structure, and intracellular transport. These identified proteins may provide information about disease pathophysiology and can serve as potential targets for therapeutic intervention of T2D. Further studies on the changes of protein expression under high glucose concentration and supplementation with vitamin D will lead to better understanding of the molecular mechanisms of T2D.

A new approach to assess the effect of photodamage on corneocyte envelope maturity using combined hydrophobicity and mechanical fragility assays.

The maturity of the corneocyte envelope (CE) provides information about the barrier functionality of the stratum corneum (SC). Corneocytes are enclosed by the CE, a protein-lipid matrix, contributing to mechanical resistance and hydrophobicity of the SC. The aim of the work was to develop a novel and robust approach to characterize CE maturity based on rigidity, hydrophobicity and surface area. This offers an alternative approach to the Nile red staining and antigenicity of involucrin to characterize the CE. The photoexposed (PE) cheek and photoprotected (PP) post-auricular sites were selected for investigation. Nine tape strips were obtained from the cheek and post-auricular sites of healthy Caucasians. CEs on the first and last tape strip were subjected to sonication to assess rigidity, and Nile red staining to determine hydrophobicity per unit surface area. In addition, the presence of involucrin and lipids was assessed to determine CE maturity by examination of the red/green pixel ratio, percentage of involucrin expressing CEs and alternatively the ratio of fluorescence density. The CE rigidity was lower in the deeper SC layers of the cheek, whereas post-auricular CEs were mechanically more resistant. Post-auricular CEs from the superficial SC had a larger surface area with a stronger fluorescence signal than those from the cheek. Interestingly, those CEs from the deeper SC layers had similar surface areas in both anatomical sites but were significantly different in hydrophobicity. These three parameters can be summarized as a relative CE maturity index that expresses CE maturity more precisely with a higher sensitivity than the conventional involucrin and Nile red staining approach. CEs of the cheek surface are more mature than CEs in the deeper SC layer, whereas CEs obtained from the post-auricular surface are more mature than those from the cheek surface. The combined method developed allows characterization of CE maturity based on hydrophobicity per unit surface area and rigidity rather than a simple ratio of lipid to involucrin. A more robust and sensitive measurement has therefore been developed addressing the limitations of earlier protocols.

Polystyrene-impregnated paper substrates for direct mass spectrometric analysis of proteins and peptides in complex matrices

Direct analysis of proteins and peptides in complex matrices is of significant importance for biological, medicinal and toxicological studies.

Discovery of noncanonical translation initiation sites through mass spectrometric analysis of protein N termini.

Translation initiation generally occurs at AUG codons in eukaryotes, although it has been shown that non-AUG or noncanonical translation initiation can also occur. However, the evidence for noncanonical translation initiation sites (TISs) is largely indirect and based on ribosome profiling (Ribo-seq) studies. Here, using a strategy specifically designed to enrich N termini of proteins, we demonstrate that many human proteins are translated at noncanonical TISs. The large majority of TISs that mapped to 5′ untranslated regions were noncanonical and led to N-terminal extension of annotated proteins or translation of upstream small open reading frames (uORF). It has been controversial whether the amino acid corresponding to the start codon is incorporated at the TIS or methionine is still incorporated. We found that methionine was incorporated at almost all noncanonical TISs identified in this study. Comparison of the TISs determined through mass spectrometry with ribosome profiling data revealed that about two-thirds of the novel annotations were indeed supported by the available ribosome profiling data. Sequence conservation across species and a higher abundance of noncanonical TISs than canonical ones in some cases suggests that the noncanonical TISs can have biological functions. Overall, this study provides evidence of protein translation initiation at noncanonical TISs and argues that further studies are required for elucidation of functional implications of such noncanonical translation initiation.

Top-down/Bottom-up Mass Spectrometry Workflow Using Dissolvable Polyacrylamide Gels.

Biologists' preeminent toolbox for separating, analyzing, and visualizing proteins is SDS-PAGE, yet recovering the proteins embedded in these polyacrylamide media as intact species is a long-standing challenge for mass spectrometry. In conventional workflows, protein mixtures from crude biological samples are electrophoretically separated at high-resolution within N,N'-methylene-bis-acrylamide cross-linked polyacrylamide gels to reduce sample complexity and facilitate sensitive characterization. However, low protein recoveries, especially for high molecular weight proteins, often hinder characterization by mass spectrometry. We describe a workflow for top-down/bottom-up mass spectrometric analyses of proteins in polyacrylamide slab gels using dissolvable, bis-acryloylcystamine-cross-linked polyacrylamide, enabling high-resolution protein separations while recovering intact proteins over a broad size range efficiently. The inferior electrophoretic resolution long associated with reducible gels has been overcome, as demonstrated by SDS-PAGE of crude tissue extracts. This workflow elutes intact proteins efficiently, supporting MS and MS/MS from proteins resolved on biologists' preferred separation platform.

Mass Spectrometric Analysis of Proteins.

Mass spectrometry is a generic technique for the structural and functional analysis of purified proteins. Instrument capabilities and the possibilities of intact protein, peptide fragmentation and native analyses are discussed. Detailed experimental protocols are described for the most commonly applied techniques of protein identification, posttranslational modification (PTM) characterization, PTM mapping, native mass spectrometry, and analysis of membrane proteins using electrospray mass spectrometry.

Biomarkers discovery by peptide and protein profiling in biological fluids based on functionalized magnetic beads purification and mass spectrometry.

Biomarkers discovery by peptide and protein profiling in biological fluids based on functionalized magnetic beads purification and mass spectrometry.