Accurate Label-Free Protein Quantitation with High- and Low-Resolution Mass Spectrometers

Krey, Jocelyn F.; Wilmarth, Phillip A.; Shin, Jung Bum; Klimek, John; Sherman, Nicholas E.; Jeffery, Erin; Choi, Dongseok; David, Larry L.; Barr-Gillespie, Peter G.

doi:10.1021/pr401017h

Cited by 127 publications

(141 citation statements)

References 33 publications

Supporting

Mentioning

139

Contrasting

Order By: Relevance

“…Afterward, the intensities of the three (or fewer) peptides with the highest intensities were averaged for every protein detected. Next, a normalized abundance factor (rTop3) was calculated by dividing the Top3 value by the sum of all Top3 values of the quantified proteins in each experiment (65), excluding contaminants. Additionally, the proteins were ranked according to their maximal rTop3 value, as calculated across each host-plant specific sample.…”

Section: Establishment Of T Urticae Lines On Different Host Plants-tmentioning

confidence: 99%

“…Subsequently we used the normalized (relative) abundance factor rTop3, based on MS1 intensity, for abundance ranking of these putative salivary proteins. This rTop3 value has been shown to correlate with the mole fraction of the protein of interest (65,90). Proteins with a high rTop3 factor are therefore assumed to be more prominent in T. urticae saliva.…”

Section: T Urticae Secretes Proteins In Artificial Diet Which Canmentioning

confidence: 99%

See 1 more Smart Citation

The Salivary Protein Repertoire of the Polyphagous Spider Mite Tetranychus urticae: A Quest for Effectors

Jonckheere

Dermauw

Zhurov

et al. 2016

Molecular & Cellular Proteomics

127

View full text Add to dashboard Cite

The two-spotted spider mite Tetranychus urticae is an extremely polyphagous crop pest. Alongside an unparalleled detoxification potential for plant secondary metabolites, it has recently been shown that spider mites can attenuate or even suppress plant defenses. Salivary constituents, notably effectors, have been proposed to play an important role in manipulating plant defenses and might determine the outcome of plant-mite interactions.Here, the proteomic composition of saliva from T. urticae lines adapted to various host plants-bean, maize, soy, and tomato-was analyzed using a custom-developed feeding assay coupled with nano-LC tandem mass spectrometry. About 90 putative T. urticae salivary proteins were identified. Many are of unknown function, and in numerous cases belonging to multimembered gene families. RNAseq expression analysis revealed that many genes coding for these salivary proteins were highly expressed in the proterosoma, the mite body region that includes the salivary glands. A subset of genes encoding putative salivary proteins was selected for whole-mount in situ hybridization, and were found to be expressed in the anterior and dorsal podocephalic glands. Strikingly, host plant dependent expression was evident for putative salivary proteins, and was further studied in detail by micro-array based genome-wide expression profiling. This meta-analysis revealed for the first time the salivary protein repertoire of a phytophagous chelicerate. The availability of this salivary proteome will assist in unraveling the molecular interface between phytophagous mites and their host plants, and may ultimately facilitate the development of mite-resistant crops. Furthermore, the technique used in this study is a time-and resourceefficient method to examine the salivary protein composition of other small arthropods for which saliva or salivary glands cannot be isolated easily. The family of spider mites (Chelicerata: Acari: Tetranychidae) comprises well over 1000 species, including several that are important pests on crops, and about 0.9 billion euro is being spent annually for their control worldwide (1, 2). These minute herbivores-about 0.5 mm in size-use their stylets to pierce leaf mesophyll cells and to inject saliva, after which they suck out the cytoplasm. This results in cell death visible as chlorotic spots sometimes accompanied by necrosis, and ultimately in leaf abscission (3,4). Among the spider mites, the From the ‡Laboratory

show abstract

Section: Establishment Of T Urticae Lines On Different Host Plants-tmentioning

confidence: 99%

Section: T Urticae Secretes Proteins In Artificial Diet Which Canmentioning

confidence: 99%

The Salivary Protein Repertoire of the Polyphagous Spider Mite Tetranychus urticae: A Quest for Effectors

Jonckheere

Dermauw

Zhurov

et al. 2016

Molecular & Cellular Proteomics

127

View full text Add to dashboard Cite

show abstract

“…(J-L) Linear regressions between relative protein amounts based on 2D-PAGE analysis of E. coli proteins 42 and the absolute proteins amounts derived from dNSAF cLL and dNIAF cLL values (Table S3B) measured in the LTQ (J), Velos (K) and Orbitrap (L) datasets. 39 We next assessed the agreement between the protein amounts quantified by dNSAF cLL , dNAAF cLL and dNIAF cLL and previously reported literature values. Out of the 1061 S. cerevisiae proteins we absolutely quantified, the abundance of 895 of these had also been measured by quantitative Western blotting of TAP-tagged proteins, 40 while the abundance of 12 of these proteins had been reported by Single Reaction Monitoring (SRM) 41 as well as quantitative Western blotting 40 (Table S3A).…”

Section: Resultsmentioning

confidence: 93%

“…Knowing that 31.3µg of E. coli extract were present in each sample 39 and summing up the absolute amount we calculated from each protein, we obtained total protein recovery rates between 47% and 68% (Table S3B). Such recoveries were respectable yet lower than what we observed from the yeast analysis, most likely because the E. coli proteins were separated on SDS-PAGE and in-gel digested prior to LC-MS/MS analyses.…”

Section: Resultsmentioning

confidence: 99%

Improving Label-Free Quantitative Proteomics Strategies by Distributing Shared Peptides and Stabilizing Variance

et al. 2015

View full text Add to dashboard Cite

In a previous study, we demonstrated that spectral counts-based label-free proteomic quantitation could be improved by distributing peptides shared between multiple proteins. Here, we compare four quantitative proteomic approaches, namely, the normalized spectral abundance factor (NSAF), the normalized area abundance factor (NAAF), normalized parent ion intensity abundance factor (NIAF), and the normalized fragment ion intensity abundance factor (NFAF). We demonstrate that label-free proteomic quantitation methods based on chromatographic peak area (NAAF), parent ion intensity in MS1 (NIAF), and fragment ion intensity (NFAF) are also improved when shared peptides are distributed on the basis of peptides unique to each isoform. To stabilize the variance inherent to label-free proteomic quantitation data sets, we use cyclic-locally weighted scatter plot smoothing (LOWESS) and linear regression normalization (LRN). Again, all four methods are improved when cyclic-LOWESS and LRN are applied to reduce variation. Finally, we demonstrate that absolute quantitative values may be derived from label-free parameters such as spectral counts, chromatographic peak area, and ion intensity when using spiked-in proteins of known amounts to generate standard curves.

show abstract

“…Label free approaches are popular due to limited sample pre-processing requirements prior to analysis compared to label based methodologies. Examples include Intensity-Based Absolute Quantification (iBAQ) and Absolute Protein Expression (APEX), which have been compared for different sample types and MS platforms [16][17][18].…”

Section: Approaches In Proteomicsmentioning

confidence: 99%

Advances in proteomics for production strain analysis

Landels

Evans

Noirel

et al. 2015

Current Opinion in Biotechnology

View full text Add to dashboard Cite

Highlights Proteomics is widely used in production strain analysis  The value of specific strategies is discussed with reference to case studies  Methodologies often based on prior application to eukaryotic systems  New developments target quantitative accuracy and proteome coverage AbstractProteomics is the large-scale study and analysis of proteins, directed to analysing protein function in a cellular context. Since the vast majority of the processes occurring in a living cell rely on protein activity, proteomics offer a unique vantage point from which researchers can dissect, characterise, understand and manipulate biological systems. When developing a production strain, proteomics offers a versatile toolkit of analytical techniques. In this commentary, we highlight a number of recent developments in this field using three industrially relevant case studies: targeted proteomic analysis of heterologous pathways in E. coli, biofuel production in Synechocystis PCC6803 and proteomic investigations of lignocellulose degradation. We conclude by discussing future developments in proteomics that will impact upon metabolic engineering and process monitoring of bio-producer strains.

show abstract

Accurate Label-Free Protein Quantitation with High- and Low-Resolution Mass Spectrometers

Cited by 127 publications

References 33 publications

The Salivary Protein Repertoire of the Polyphagous Spider Mite Tetranychus urticae: A Quest for Effectors

The Salivary Protein Repertoire of the Polyphagous Spider Mite Tetranychus urticae: A Quest for Effectors

Improving Label-Free Quantitative Proteomics Strategies by Distributing Shared Peptides and Stabilizing Variance

Advances in proteomics for production strain analysis

Contact Info

Product

Resources

About