Thin Thin Aye

Triplex protein quantification based on stable isotope labeling by peptide dimethylation applied to cell and tissue lysates

Stable isotope labeling is at present one of the most powerful methods in quantitative proteomics. Stable isotope labeling has been performed at both the protein as well as the peptide level using either metabolic or chemical labeling. Here, we present a straightforward and cost‐effective triplex quantification method that is based on stable isotope dimethyl labeling at the peptide level. Herein, all proteolytic peptides are chemically labeled at their α‐ and ϵ‐amino groups. We use three different isotopomers of formaldehyde to enable the parallel analysis of three different samples. These labels provide a minimum of 4 Da mass difference between peaks in the generated peptide triplets. The method was evaluated based on the quantitative analysis of a cell lysate, using a typical “shotgun” proteomics experiment. While peptide complexity was increased by introducing three labels, still more than 1300 proteins could be identified using 60 μg of starting material, whereby more than 600 proteins could be quantified using at least four peptides per protein. The triplex labeling was further utilized to distinguish specific from aspecific cAMP binding proteins in a chemical proteomics experiment using immobilized cAMP. Thereby, differences in abundance ratio of more than two orders of magnitude could be quantified.

Proteomics of human cerebrospinal fluid: Discovery and verification of biomarker candidates in neurodegenerative diseases using quantitative proteomics

There is an urgent need for novel biomarkers that can be used to improve the diagnosis, predict the disease progression, improve our understanding of the pathology or serve as therapeutic targets for neurodegenerative diseases. Cerebrospinal fluid (CSF) is in direct contact with the CNS and reflects the biochemical state of the CNS under different physiological and pathological settings. Because of this, CSF is regarded as an excellent source for identifying biomarkers for neurological diseases and other diseases affecting the CNS. Quantitative proteomics and sophisticated computational software applied to analyze the protein content of CSF has been fronted as an attractive approach to find novel biomarkers for neurological diseases. This review will focus on some of the potential pitfalls in biomarker studies using CSF, summarize the status of the field of CSF proteomics in general, and discuss some of the most promising proteomics biomarker study approaches. A brief status of the biomarker discovery efforts in multiple sclerosis, Alzheimers disease, and Parkinsons disease is also given.

An Augmented Multiple-Protease-Based Human Phosphopeptide Atlas

Although mass-spectrometry-based screens enable thousands of protein phosphorylation sites to be monitored simultaneously, they often do not cover important regulatory sites. Here, we hypothesized that this is due to the fact that nearly all large-scale phosphoproteome studies are initiated by trypsin digestion. We tested this hypothesis using multiple proteases for protein digestion prior to Ti(4+)-IMAC-based enrichment. This approach increases the size of the detectable phosphoproteome substantially and confirms the considerable tryptic bias in public repositories. We define and make available a less biased human phosphopeptide atlas of 37,771 unique phosphopeptides, correlating to 18,430 unique phosphosites, of which fewer than 1/3 were identified in more than one protease data set. We demonstrate that each protein phosphorylation site can be linked to a preferred protease, enhancing its detection by mass spectrometry (MS). For specific sites, this approach increases their detectability by more than 1,000-fold.

Selectivity in Enrichment of cAMP-dependent Protein Kinase Regulatory Subunits Type I and Type II and Their Interactors Using Modified cAMP Affinity Resins

cAMP regulates cellular functions primarily by activating PKA. The involvement of PKAs in various signaling pathways occurring simultaneously in different cellular compartments necessitates stringent spatial and temporal regulation. This specificity is largely achieved by binding of PKA to protein scaffolds, whereby a distinct group of proteins called A kinase anchoring proteins (AKAPs) play a dominant role. AKAPs are a diverse family of proteins that all bind via a small PKA binding domain to the regulatory subunits of PKA. The binding affinities between PKA and several AKAPs can be different for different isoforms of the regulatory subunits of PKA. Here we employ a combination of affinity chromatography and mass spectrometry-based quantitative proteomics to investigate specificity in PKA-AKAP interactions. Three different immobilized cAMP analogs were used to enrich for PKA and its interacting proteins from several systems; HEK293 and RCC10 cells and rat lung and testis tissues. Stable isotope labeling was used to confidently identify and differentially quantify target proteins and their preferential binding affinity for the three different cAMP analogs. We were able to enrich all four isoforms of the regulatory subunits of PKA and concomitantly identify more than 10 AKAPs. A selective enrichment of the PKA RI isoforms could be achieved; which allowed us to unravel which AKAPs bind preferentially to the RI or RII regulatory domains of PKA. Of the twelve AKAPs detected, seven preferentially bound to RII, whereas the remaining five displayed at least dual specificity with a potential preference for RI. For some of these AKAPs our data provide the first insights into their specificity.

Use of stable isotope dimethyl labeling coupled to selected reaction monitoring to enhance throughput by multiplexing relative quantitation of targeted proteins

In this manuscript, we present a proof-of-concept study for targeted relative protein quantitation workflow using chemical labeling in the form of dimethylation, coupled with selected reaction monitoring (dimethyl-SRM). We first demonstrate close to complete isotope incorporation for all peptides tested. The accuracy, reproducibility, and linear dynamic range of quantitation are further assessed based on known ratios of nonhuman standard proteins spiked into human cerebrospinal fluid (CSF) as a model complex matrix. Quantitation reproducibility below 20% (CV < 20%) was obtained for analyte concentrations present at a dynamic range of 4 orders of magnitude lower than that of the background proteins. An error of less than 15% was observed when measuring the abundance of 44 out of 45 major human plasma proteins. Dimethyl-SRM was further examined by comparing the relative quantitation of eight proteins in human CSF with the relative quantitation obtained using synthetic heavy peptides coupled to stable isotope dilution-SRM (SID-SRM). Comparison between the two methods reveals that the correlation between dimethyl-SRM and SID-SRM is within 0.3-33% variation, demonstrating the accuracy of relative quantitation using dimethyl-SRM. Dimethyl labeling coupled with SRM provides a fast, convenient, and cost-effective alternative for relative quantitation of a large number of candidate proteins/peptides.

Protein profiling reveals inter-individual protein homogeneity of arachnoid cyst fluid and high qualitative similarity to cerebrospinal fluid

BackgroundThe mechanisms behind formation and filling of intracranial arachnoid cysts (AC) are poorly understood. The aim of this study was to evaluate AC fluid by proteomics to gain further knowledge about ACs. Two goals were set: 1) Comparison of AC fluid from individual patients to determine whether or not temporal AC is a homogenous condition; and 2) Evaluate the protein content of a pool of AC fluid from several patients and qualitatively compare this with published protein lists of cerebrospinal fluid (CSF) and plasma.MethodsAC fluid from 15 patients with temporal AC was included in this study. In the AC protein comparison experiment, AC fluid from 14 patients was digested, analyzed by LC-MS/MS using a semi-quantitative label-free approach and the data were compared by principal component analysis (PCA) to gain knowledge of protein homogeneity of AC. In the AC proteome evaluation experiment, AC fluid from 11 patients was pooled, digested, and fractionated by SCX chromatography prior to analysis by LC-MS/MS. Proteins identified were compared to published databases of proteins identified from CSF and plasma. AC fluid proteins not found in these two databases were experimentally searched for in lumbar CSF taken from neurologically-normal patients, by a targeted protein identification approach called MIDAS (Multiple Reaction Monitoring (MRM) initiated detection and sequence analysis).ResultsWe did not identify systematic trends or grouping of data in the AC protein comparison experiment, implying low variability between individual proteomic profiles of AC.In the AC proteome evaluation experiment, we identified 199 proteins. When compared to previously published lists of proteins identified from CSF and plasma, 15 of the AC proteins had not been reported in either of these datasets. By a targeted protein identification approach, we identified 11 of these 15 proteins in pooled CSF from neurologically-normal patients, demonstrating that the majority of abundant proteins in AC fluid also can be found in CSF. Compared to plasma, as many as 104 proteins in AC were not found in the list of 3017 plasma proteins.ConclusionsBased on the protein content of AC fluid, our data indicate that temporal AC is a homogenous condition, pointing towards a similar AC filling mechanism for the 14 patients examined. Most of the proteins identified in AC fluid have been identified in CSF, indicating high similarity in the qualitative protein content of AC to CSF, whereas this was not the case between AC and plasma. This indicates that AC is filled with a liquid similar to CSF. As far as we know, this is the first proteomics study that explores the AC fluid proteome.

Spatial Organization in Protein Kinase A Signaling Emerged at the Base of Animal Evolution

In phosphorylation-directed signaling, spatial and temporal control is organized by complex interaction networks that diligently direct kinases toward distinct substrates to fine-tune specificity. How these protein networks originate and evolve into complex regulatory machineries are among the most fascinating research questions in biology. Here, spatiotemporal signaling is investigated by tracing the evolutionary dynamics of each functional domain of cAMP-dependent protein kinase (PKA) and its diverse set of A-kinase anchoring proteins (AKAPs). Homologues of the catalytic (PKA-C) and regulatory (PKA-R) domains of the (PKA-R)2-(PKA-C)2 holoenzyme were found throughout evolution. Most variation was observed in the RIIa of PKA-R, crucial for dimerization and docking to AKAPs. The RIIa domain was not observed in all PKA-R homologues. In the fungi and distinct protist lineages, the RIIa domain emerges within PKA-R, but it displays large sequence variation. These organisms do not harbor homologues of AKAPs, suggesting that efficient docking to direct spatiotemporal PKA activity evolved in multicellular eukaryotes. To test this in silico hypothesis, we experimentally screened organisms with increasing complexity by cAMP-based chemical proteomics to reveal that the occurrence of PKA-AKAP interactions indeed coincided and expanded within vertebrates, suggesting a crucial role for AKAPs in the advent of metazoan multicellularity.

Probing the Specificity of Protein–Protein Interactions by Quantitative Chemical Proteomics

Chemical proteomics is a versatile tool to investigate protein-small molecule interactions, but can be extended to probe also secondary binding investigating small molecule-protein 1-protein 2 interactions, providing insight into protein scaffolds. This application of chemical proteomics has in particular been applied extensively to cyclic nucleotide (cAMP, cGMP) signaling. cAMP regulates cellular functions primarily by activating cAMP-dependent protein kinase (PKA). Compartmentalization of PKA plays an important role in the specificity of cAMP signaling events and is mediated by interaction of the regulatory subunit (PKA-R) with A-kinase anchoring proteins (AKAPs), which often form the core of even larger protein machineries. The selective binding of AKAPs to one of the major isoforms PKA-R type I (PKA-RI) and PKA-R type II (PKA-RII) is an important feature of cAMP/PKA signaling. However, this specificity is not well established for most AKAPs. Here, we describe a chemical proteomics approach that combines cAMP-based affinity chromatography with quantitative mass spectrometry to investigate PKA-R isoform/AKAP specificity directly in lysates of cells and tissues of any origin. With this tool, several novel PKA-R/AKAP specificities can be easily resolved.