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Bentz, Maria, et al.
(author)
Membrane protein identification: N-terminal labeling of nontryptic membrane protein peptides facilitates database searching
2008
In: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 7:2, s. 659-665
Journal article (peer-reviewed) 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.
2.
Cifani, Paolo, et al.
(author)
Hunting for Protein Markers of Hypoxia by Combining Plasma Membrane Enrichment with a New Approach to Membrane Protein Analysis
2011
In: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 10:4, s. 1645-1656
Journal article (peer-reviewed) 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.
3.
Levander, Fredrik, et al.
(author)
Automated reporting from gel-based proteomics experiments using the open source Proteios database application
2007
In: Proteomics. - : Wiley. - 1615-9861 .- 1615-9853. ; 7:5, s. 668-674
Journal article (peer-reviewed) abstract
The assembly of data from different parts of proteomics workflow is often a major bottleneck in proteomics. Furthermore, there is an increasing demand for the publication of details about protein identifications due to the problems with false-positive and false-negative identifications. In this report, we describe how the open-source Proteios software has been expanded to automate the assembly of the different parts of a gel-based proteomics workflow. In Proteios it is possible to generate protein identification reports that contain all the information currently required by proteomics journals. It is also possible for the user to specify maximum allowed false positive ratios, and reports are automatically generated with the corresponding score cut-offs calculated. When protein identification is conducted using multiple search engines, the score thresholds that correlate to the predetermined error rate are also explicitly calculated for proteins that appear on the result lists of more than one search engine.
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Wåhlander, Åsa, et al.
(author)
Development of reagents for differential protein quantitation by subtractive parent (precursor) ion scanning
2007
In: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 6:3, s. 1101-1113
Journal article (peer-reviewed) abstract
We present a generic approach for quantitative differential proteomics that reduces data complexity in proteome analysis by automated selection of peptides for MS/MS analysis according to their isotope-labeling ratio. Isotopic reagents were developed that give products which fragment easily to generate a unique pair of signature ions. Using the ion-pair ratio, we show that it is possible to select only BSA peptides (with a 3:1 light heavy isotope ratio) for MS/MS when spiked in a whole yeast extract using Parent (precursor) Ion Quantitation Scanning (PIQS) for MS/MS.
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