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Low affinity and transient protein-protein interactions, such as short linear motif (SLiM)-based interactions, require dedicated experimental tools for discovery and validation. Here, we evaluated and compared biotinylated peptide pulldown and protein interaction screen on peptide matrix (PRISMA) coupled to massspectrometry (MS) using a set of peptides containing interaction motifs. Eight different peptide sequences that engage in interactions with three distinct protein domains (KEAP1 Kelch, MDM2 SWIB, and TSG101 UEV) with a wide range of affinities were tested. We found that peptide pulldown can be an effective approach for SLiM validation, however, parameters such as protein abundance and competitive interactions can prevent the capture of known interactors. The use of tandem peptide repeats improved the capture and preservation of some interactions. When testing PRISMA, it failed to provide comparable results for model peptides that successfully pulled down known interactors using biotinylated peptide pulldown. Overall, in our hands, we find that albeit more laborious, biotin-peptide pulldown was more successful in terms of validation of known interactions. Our results highlight that the tested affinity-capture MS-based methods for validation of SLiM-based interactions from cell lysates are suboptimal, and we identified parameters for consideration for method development. more...

Viral infections cause large problems in the world and deeper understanding of the disease mechanisms is needed. Here we present an analytical strategy to investigate the host cell protein changes during human adenovirus type 2 (HAdV-C2 or Ad2) infection of lung fibro-blasts by stable isotope labelling of amino acids in cell culture (SILAC) and nanoLC-MS/MS. This work focuses on early phase of infection (6 and 12 h post-infection (hpi)) but the data is combined with previously published late phase (24 and 36 hpi) proteomics data to produce a time series covering the complete infection. As many as 2169 proteins were quantitatively monitored from 6 to 36 hpi, while some proteins were time-specific. After applying different filter criteria, 2027 and 2150 proteins were quantified at 6 and 12 hpi and among them, 431 and 544 were significantly altered at the two time points. Pathway analysis showed that the De novo purine and pyrimidine biosynthesis, Glycolysis and Cytoskeletal regulation by Rho GTPase pathways were activated early during infection while inactivation of the Integrin signalling pathway started between 6 and 12 hpi. Moreover, upstream regulator analysis predicted MYC to be activated with time of infection and protein and RNA data for genes controlled by this transcription factor showed good correlation, which validated the use of protein data for this prediction. Among the identified phosphorylation sites, a group related to glycolysis and cytoskeletal reorganization were up-regulated during infection. The results show specific aspects on how the host cell proteins, the final products in the genetic information flow, are influenced by Ad2 infection, which would be overlooked if only knowledge derived from mRNA data is considered. more...

Mass spectrometry-based proteomics benefits from efficient digestion of protein samples. In this study, trypsin was immobilized on nanoporous anodized alumina membranes to create an enzyme reactor suitable for peptide mass fingerprinting. The membranes were derivatized with 3-aminopropyltriethoxysilane and the amino groups were activated with carbonyldiimidazole to allow coupling of porcine trypsin via c-amino groups. The function was assessed using the artificial substrate Na-Benzoyl-L-arginine 4-nitroanilide hydrochloride, bovine ribonuclease A and a human plasma sample. A 10-membrane flow-through reactor was used for fragmentation and MS analysis after a single pass of substrate both by collection of product and subsequent off-line analysis, and by coupling on-line to the instrument. The peptide pattem allowed correct identification of the single target protein in both cases, and of > 70 plasma proteins in single pass mode followed by LC-MS analysis. The reactor retained 76% of the initial activity after 14 days of storage and repeated use at room temperature. Significance: This manuscript describes the design of a stable enzyme reactor that allows efficient and fast digestion with negligible leakage of enzyme and enzyme fragments. The high stability facilitates the use in an online-setup with MS detection since it allows the processing of multiple samples within an extended period of time without replacement., QC 20180216 more...

A deeper understanding of how viruses reprogramtheir hosts for production of progeny is needed to combatinfections. Most knowledge on the regulation of cellular geneexpression during adenovirus infection is derived from mRNAstudies. Here, we investigated the changes in protein expressionduring the late phase of adenovirus type 2 (Ad2) infection of theIMR-90 cell line by stable isotope labeling in cell culture withsubsequent liquid chromatography−high resolution tandemmass spectrometric analysis. Two biological replicates of samplescollected at 24 and 36 h post-infection (hpi) were investigated using swapped labeling. In total, 2648 and 2394 proteins werequantified at 24 and 36 hpi, respectively. Among them, 659 and 645 were deregulated >1.6-fold at the two time points. Theprotein expression was compared with RNA expression using cDNA sequencing data. The correlation was surprisingly low(r = 0.3), and several examples of posttranscriptional regulation were observed; e.g., proteins related to carbohydrate metabolismwere up-regulated at the protein level but unchanged at the RNA level, whereas histone proteins were down-regulated at theprotein level but up-regulated at the RNA level. The deregulation of cellular gene expression by adenovirus is mediated atmultiple levels and more complex than hitherto believed. more...

Background: Tyrosine phosphorylation (pTyr) is an important cancer relevant posttranslational modification since it regulates protein activity and cellular localization. By controlling cell growth and differentiation it plays an important role in tumor development. This paper describes a novel approach for detection and visualization of a panel of pTyr proteins in tumors using in situ proximity ligation assay. Methods: K562 leukemia cells were treated with tyrosine kinase and/or phosphatase inhibitors to induce differences in pTyr levels and mimic cells with different malignant properties. Cells were then probed with one antibody against the pTyr modification and another probe against the detected protein, resulting in a detectable fluorescent signal once the probes were in proximity. Results: Total and protein specific pTyr levels on ABL, SHC, ERK2 and PI3K proteins were detected and samples of control and treated cells were distinguished at the pTyr level using this novel approach. Promising results were also detected for formalin fixed and paraffin embedded cells in the micro array format. Conclusions: This application of in situ proximity ligation assay is valuable in order to study the pTyr modification of a panel of proteins in large data sets to validate mass spectrometric data and to be combined with tissue microarrays. The approach offers new opportunities to reveal the pTyr signatures in cells of different malignant properties that can be used as biomarker of disease in the future. more...

Analysis of proteins and their posttranslational modifications is important for understanding different biological events. For analysis of viral proteomes, an optimal protocol includes production of a highly purified virus that can be investigated with a high-resolving analytical method. In this Methods in Molecular Biology paper we describe a working strategy for how structural proteins in the Adenovirus particle can be studied using liquid chromatography–high-resolving mass spectrometry. This method provides information on the chemical composition of the virus particle. Further, knowledge about amino acids carrying modifications that could be essential for any part of the virus life cycle is collected. We describe in detail alternatives available for preparation of virus for proteome analysis as well as choice of mass spectrometric instrumentation suitable for this kind of analysis. more...

We have combined 2-D SOS-PAGE with liquid chromatography-high resolving mass spectrometry (LC-MS) to explore the proteome of the adenovirus type 2 (Ad2) at the level of post translational modifications (PTMs). The experimental design included in-solution digestion, followed by titanium dioxide enrichment, as well as in-gel digestion of polypeptides after separation of Ad2 capsid proteins by 1-D and 2-D SOS-PAGE. All samples were analyzed using LC-MS with subsequent manual verification of PTM positions. The results revealed new phosphorylation sites that can explain the observed trains of protein spots observed for the pIII, pIIIa and ply proteins. The pin protein was found to be the most highly modified protein with now 18 verified sites of phosphorylation, three sites of nitrated tyrosine and one sulfated tyrosine. Nitrated tyrosines were also identified in pII. Lysine acetylations were detected in pII and pVI. The findings make the Ad2 virion much more complex than hitherto believed. more...

Nonmuscle invasive tumors of the bladder often recur and thereby bladder cancer patients need regular re-examinations which are invasive, unpleasant, and expensive. A noninvasive and less expensive method, e.g. a urine dipstick test, for monitoring recurrence would thus be advantageous. In this study, the complementary techniques mass spectrometry (MS) and Western blotting (WB)/dot blot (DB) were used to screen the urine samples from bladder cancer patients. High resolving MS was used to analyze and quantify the urinary proteome and 29 proteins had a significantly higher abundance (p<0.05) in bladder cancer samples compared with control urine samples. The increased abundance found in urine from bladder cancer patients compared with controls was confirmed with Western blot for four selected proteins; fibrinogen β chain precursor, apolipoprotein E, α-1-antitrypsin, and leucine-rich α-2-glycoprotein 1. Dot blot analysis of an independent urine sample set pointed out fibrinogen β chain and α-1-antitrypsin as most interesting biomarkers having sensitivity and specificity values in the range of 66-85%. Exploring the Human Protein Atlas (HPA) also revealed that bladder cancer tumors are the likely source of these proteins. They have the potential of being useful in diagnosis, monitoring of recurrence and thus may improve the treatment of bladder tumors, especially nonmuscle invasive tumors. more...

Phosphorylation of protein tyrosine residues regulates many cell functions and has also been proved to be involved in oncogenesis. Thus, the identification of the phosphotyrosine (pTyr) proteome of cells is a very important task. Since tyrosine phosphorylation represents only around 1% of the total human phosphoproteome, the study of pTyr proteins is rather challenging. Here we report the optimization study of the phosphotyrosine proteome using K562 cells as a model system. A substantial segment of the phosphotyrosine proteome of K562 cells was characterized by immunoaffinity enrichment with 4G10 and PYKD1 antibodies followed by LC-MS/MS analysis. 480 non-redundant pTyr peptides corresponding to 342 pTyr proteins were found. 141 pTyr peptides were not described elsewhere. The mass spectrometry approach involving high-resolving FTMS analysis of precursor ions and subsequent detection of CID fragments in a linear ion trap was considered as optimal. For detection of low abundant pTyr peptides pooling of individual immunoaffinity enrichments for one LC-MS/MS analysis was crucial. The enrichment properties of the monoclonal PYKD1 antibody were presented for the first time, also in comparison to the 4G10 antibody. PYKD1 was found to be more effective for protein enrichment (1.2 and 5% efficiency at peptide and protein level correspondingly), while 4G10 showed better results when peptide enrichment was performed (15% efficiency versus 3.6% at protein level). Substantially different subsets of the phosphoproteome were enriched by these antibodies. This finding together with previous studies demonstrates that comprehensive pTyr proteome characterization by immunoprecipitation requires multiple antibodies to be used for the affinity enrichment. more...

We describe fluorescence-based 2-D gel electrophoresis methods for visualization of low abundant cancer relevant tyrosine phosphorylated (pTyr) proteins The methods investigated were fluorescent Western blotting and two-dimensional difference gel electrophoresis (2-D DIGE) for detection of non-enriched and immunoaffinity enriched pTyr protein patterns The same anti-phosphotyrosine specific antibody 4G10 was used for both approaches The results from fluorescent Western blotting of total proteins and from enriched CyDye DIGE pre-labeled pTyr proteins showed similar down regulation of phosphorylation upon treating of cells from a cancer model system (K562 chronic myeloid leukemia cells) with imatinib This treatment introduced a known perturbation of phosphorylation that enabled testing of these new approaches to analyze variations in tyrosine phosphorylation levels Enrichment of pTyr proteins was found highly advantageous for the outcome Out of a simplified 2 D DIGE experiment of immunoaffinity enriched control and treated pTyr proteins differential analysis as well as protein identification by mass spectrometry (MS) was possible. more...