1.
Antberg, Linn, et al.
(författare)
Critical Comparison of Multidimensional Separation Methods for Increasing Protein Expression Coverage
2012
Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 11:5, s. 2644-2652
Tidskriftsartikel (refereegranskat) abstract
We present a comparison of two-dimensional separation methods and how they affect the degree of coverage of protein expression in complex mixtures. We investigated the relative merits of various protein and peptide separations prior to acidic reversed-phase chromatography directly coupled to an ion trap mass spectrometer. The first dimensions investigated were density gradient organelle fractionation of cell extracts, 1D SDS-PAGE protein separation followed by digestion by trypsin or GluC proteases, strong cation exchange chromatography, and off-gel isoelectric focusing of tryptic peptides. The number of fractions from each first dimension and the total data accumulation RP-HPLC-MS/MS time was kept constant and the experiments were run in triplicate. We find that the most critical parameters are the data accumulation time, which defines the level of under-sampling and the avoidance of peptides from high expression level proteins eluting over the entire gradient.
2.
Bendz, Maria, et al.
(författare)
Quantification of Membrane Proteins Using Nonspecific Protease Digestions
2009
Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 8:12, s. 5666-5673
Tidskriftsartikel (refereegranskat) abstract
We present a mass spectrometry-based method for the identification and quantification of membrane proteins using the low-specificity protease Proteinase K, at very high pH, to digest proteins isolated by a modified SDS-PAGE protocol. The resulting peptides are modified with a fragmentation-directing isotope labeled tag. We apply the method to quantify differences in membrane protein expression of Bacillus subtilis grown in the presence or absence of glucose.
3.
Bentz, Maria, et al.
(författare)
Membrane protein identification: N-terminal labeling of nontryptic membrane protein peptides facilitates database searching
2008
Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 7:2, s. 659-665
Tidskriftsartikel (refereegranskat) abstract
Membrane proteins are fairly refractory to digestion especially by trypsin, and less specific proteases, such as elastase and pepsin, are much more effective. However, database searching using nontryptic peptides is much less effective because of the lack of charge localization at the N and C termini and the absence of sequence specificity. We describe a method for N-terminal-specific labeling of peptides from nontryptic digestions of membrane proteins, which facilitates Mascot database searching and can be used for relative quantitation. The conditions for digestion have been optimized to obtain peptides of a suitable length for mass spectrometry (MS) fragmentation. We show the effectiveness of the method using a plasma membrane preparation from a leukemia cell line and demonstrate a large increase in the number of membrane proteins, with small extra-membranar domains being identified in comparison to previous published methods.
4.
Burra, Dharani, et al.
(författare)
Phosphite-induced changes of the transcriptome and secretome in Solanum tuberosum leading to resistance against Phytophthora infestans
2014
Ingår i: BMC Plant Biology. - : Springer Science and Business Media LLC. - 1471-2229. ; 14
Tidskriftsartikel (refereegranskat) abstract
Background: Potato late blight caused by the oomycete pathogen Phytophthora infestans can lead to immense yield loss. We investigated the transcriptome of Solanum tubersoum (cv. Desiree) and characterized the secretome by quantitative proteomics after foliar application of the protective agent phosphite. We also studied the distribution of phosphite in planta after application and tested transgenic potato lines with impaired in salicylic and jasmonic acid signaling. Results: Phosphite had a rapid and transient effect on the transcriptome, with a clear response 3 h after treatment. Strikingly this effect lasted less than 24 h, whereas protection was observed throughout all time points tested. In contrast, 67 secretome proteins predominantly associated with cell-wall processes and defense changed in abundance at 48 h after treatment. Transcripts associated with defense, wounding, and oxidative stress constituted the core of the phosphite response. We also observed changes in primary metabolism and cell wall-related processes. These changes were shown not to be due to phosphate depletion or acidification caused by phosphite treatment. Of the phosphite-regulated transcripts 40% also changed with beta-aminobutyric acid (BABA) as an elicitor, while the defence gene PR1 was only up-regulated by BABA. Although phosphite was shown to be distributed in planta to parts not directly exposed to phosphite, no protection in leaves without direct foliar application was observed. Furthermore, the analysis of transgenic potato lines indicated that the phosphite-mediated resistance was independent of the plant hormones salicylic and jasmonic acid. Conclusions: Our study suggests that a rapid phosphite-triggered response is important to confer long-lasting resistance against P. infestans and gives molecular understanding of its successful field applications.
5.
Cappadona, Salvatore, et al.
(författare)
Improved label-free LC-MS analysis by wavelet-based noise rejection
2010
Ingår i: Journal of Biomedicine and Biotechnology. - : Hindawi Limited. - 1110-7251 .- 1110-7243. ; 2010
Tidskriftsartikel (refereegranskat) abstract
Label-free LC-MS analysis allows determining the differential expression level of proteins in multiple samples, without the use of stable isotopes. This technique is based on the direct comparison of multiple runs, obtained by continuous detection in MS mode. Only differentially expressed peptides are selected for further fragmentation, thus avoiding the bias toward abundant peptides typical of data-dependent tandem MS. The computational framework includes detection, alignment, normalization and matching of peaks across multiple sets, and several software packages are available to address these processing steps. Yet, more care should be taken to improve the quality of the LC-MS maps entering the pipeline, as this parameter severely affects the results of all downstream analyses. In this paper we show how the inclusion of a preprocessing step of background subtraction in a common laboratory pipeline can lead to an enhanced inclusion list of peptides selected for fragmentation and consequently to better protein identification.
6.
Chawade, Aakash, et al.
(författare)
Data Processing Has Major Impact on the Outcome of Quantitative Label-Free LC-MS Analysis
2015
Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 14:2, s. 676-687
Tidskriftsartikel (refereegranskat) abstract
High-throughput multiplexed protein quantification using mass spectrometry is steadily increasing in popularity, with the two major techniques being data-dependent acquisition (DDA) and targeted acquisition using selected reaction monitoring (SRM). However, both techniques involve extensive data processing, which can be performed by a multitude of different software solutions. Analysis of quantitative LC-MS/MS data is mainly performed in three major steps: processing of raw data, normalization, and statistical analysis. To evaluate the impact of data processing steps, we developed two new benchmark data sets, one each for DDA and SRM, with samples consisting of a long-range dilution series of synthetic peptides spiked in a total cell protein digest. The generated data were processed by eight different software workflows and three postprocessing steps. The results show that the choice of the raw data processing software and the postprocessing steps play an important role in the final outcome. Also, the linear dynamic range of the DDA data could be extended by an order of magnitude through feature alignment and a charge state merging algorithm proposed here. Furthermore, the benchmark data sets are made publicly available for further benchmarking and software developments.
7.
Chawade, Aakash, et al.
(författare)
Normalyzer: A Tool for Rapid Evaluation of Normalization Methods for Omics Data Sets
2014
Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 13:6, s. 3114-3120
Tidskriftsartikel (refereegranskat) abstract
High-throughput omics data often contain systematic biases introduced during various steps of sample processing and data generation. As the source of these biases is usually unknown, it is difficult to select an optimal normalization method for a given data set. To facilitate this process, we introduce the open-source tool "Normalyzer". It normalizes the data with 12 different normalization methods and generates a report with several quantitative and qualitative plots for comparative evaluation of different methods. The usefulness of Normalyzer is demonstrated with three different case studies from quantitative proteomics and transcriptomics. The results from these case studies show that the choice of normalization method strongly influences the outcome of downstream quantitative comparisons. Normalyzer is an R package and can be used locally or through the online implementation at http://quantitativeproteomics.org/normalyzer.
8.
Cifani, Paolo, et al.
(författare)
Hunting for Protein Markers of Hypoxia by Combining Plasma Membrane Enrichment with a New Approach to Membrane Protein Analysis
2011
Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 10:4, s. 1645-1656
Tidskriftsartikel (refereegranskat) abstract
Nontransient hypoxia is strongly associated with malignant lesions, resulting in aggressive behavior and resistance to treatment. We present an analysis of mRNA and protein expression changes in neuroblastoma cell lines occurring upon the transition from normoxia to hypoxia. The correlation between mRNA and protein level changes was poor, although some known hypoxia-driven genes and proteins correlated well. We present previously undescribed membrane proteins expressed under hypoxic conditions that are candidates for evaluation as biomarkers.
9.
10.
Deutsch, Eric W., et al.
(författare)
TraML: a standard format for exchange of selected reaction monitoring transition lists
2012
Ingår i: Molecular & Cellular Proteomics. - 1535-9484. ; 11:4, s. 111-015040
Tidskriftsartikel (refereegranskat) abstract
Abstract in UndeterminedTargeted proteomics via selected reaction monitoring (SRM) is a powerful mass spectrometric technique affording higher dynamic range, increased specificity and lower limits of detection than other shotgun mass spectrometry methods when applied to proteome analyses. However, it involves selective measurement of predetermined analytes, which requires more preparation in the form of selecting appropriate signatures for the proteins and peptides that are to be targeted. There is a growing number of software programs and resources for selecting optimal transitions and the instrument settings used for the detection and quantification of the targeted peptides, but the exchange of this information is hindered by a lack of a standard format. We have developed a new standardized format, called TraML, for encoding transition lists and associated metadata. In addition to introducing the TraML format, we demonstrate several implementations across the community, and provide semantic validators, extensive documentation, and multiple example instances to demonstrate correctly written documents. Widespread use of TraML will facilitate the exchange of transitions, reduce time spent handling incompatible list formats, increase the reusability of previously optimized transitions, and thus accelerate the widespread adoption of targeted proteomics via SRM.
