Repeatability and Reproducibility in Proteomic Identifications by Liquid Chromatography−Tandem Mass Spectrometry

Tabb, David L.; Vega‐Montoto, Lorenzo; Rudnick, Paul; Variyath, Asokan Mulayath; Ham, Amy Joan L.; Bunk, David M.; Kilpatrick, Lisa E.; Billheimer, Dean; Blackman, Ronald K.; Cardasis, Helene L.; Carr, Steven A.; Clauser, Karl R.; Jaffe, Jacob D.; Kowalski, Kevin A.; Neubert, Thomas A.; Regnier, Fred E.; Schilling, Birgit; Tegeler, Tony; Wang, Mu; Wang, Pei; Whiteaker, Jeffrey R.; Zimmerman, Lisa J.; Fisher, Susan J.; Gibson, Bradford W.; Kinsinger, Christopher R.; Mesri, Mehdi; Rodriguez, Henry; Stein, Stephen E.; Tempst, Paul; Paulovich, Amanda G.; Liebler, Daniel C.; Spiegelman, Cliff

doi:10.1021/pr9006365

Cited by 509 publications

(520 citation statements)

References 34 publications

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“…We therefore compared a standard Laemmli-mediated protein extraction with TRIzol©-mediated protein extraction for EVs captured from the MCF-10A, MCF-7 and MDA-MB-231 breast cell lines, and found that 57.7% (843), 69.2% (1338) and 57.0% (543), respectively, of the identified EV proteins were common to both extraction methods. We typically observe 65–75% overlap of protein identifications over triplicate technical replicate nanoLC-MS/MS analyses using conventional Laemmli extraction, which is comparable with other published values [27]. In consideration of this technical variability of the nanoLC-MS/MS protein identification workflow, these data suggest that TRIzol© extraction of proteins from EVs yields results that are comparable with those for Laemmli extraction.…”

Section: Discussionsupporting

confidence: 86%

Proteome profiling of extracellular vesicles captured with the affinity peptide Vn96: comparison of Laemmli and TRIzol© protein‐extraction methods

Joy

Ayre

Chute

et al. 2018

J of Extracellular Vesicle

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Sample amount is often a limiting factor for multi-parametric analyses that encompass at least three areas of ‘-omics’ research: genomics, transcriptomics and proteomics. Limited sample amounts are also an important consideration when these multi-parametric analyses are performed on extracellular vesicles (EVs), as the amount of EVs (and EV cargo) that can be isolated is often very low. It is well understood that a monophasic solution of phenol and guanidine isothiocyanate (i.e. TRIzol©) can simultaneously isolate DNA, RNA and proteins from biological samples; however, it is most commonly used for the extraction of RNA. Validation of this reagent for the isolation of multiple classes of biological molecules from EVs would provide a widely applicable method for performing multi-parametric analyses of EV material. In this report, we describe a comparison of proteins identified from EVs processed with either TRIzol© or the conventional Laemmli buffer protein-extraction reagents. EVs were isolated from 3 mL of cell-culture supernatant derived from MCF-10A, MCF-7 and MDA-MB-231 cells using the Vn96 EV capture technology. For the TRIzol© extraction protocol, proteins were precipitated with acetone from the organic phase and then re-solubilized in a mixture of 8M urea, 0.2% SDS and 1 M Tris-HCl pH 6.8, followed by dilution in 5× loading buffer prior to fractionation with 1D SDS-PAGE. NanoLC-MS/MS of the trypsin-digested proteins was used to generate proteomic profiles from EV protein samples extracted with each method. Of the identified proteins, 57.7%, 69.2% and 57.0% were common to both extraction methods for EVs from MCF-10A, MCF-7 and MDA-MB-231, respectively. Our results suggest that TRIzol© extraction of proteins from EVs has significant equivalence to the traditional Laemmli method. The advantage of using TRIzol© reagent is the ability to accumulate multi-parametric data (e.g., RNA and protein profiles) on the same limited EV sample while minimizing sample preparation and processing time.

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Section: Discussionsupporting

confidence: 86%

Proteome profiling of extracellular vesicles captured with the affinity peptide Vn96: comparison of Laemmli and TRIzol© protein‐extraction methods

Joy

Ayre

Chute

et al. 2018

J of Extracellular Vesicle

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“…all 500 kinases, 1400 transcription factors, etc.). Likewise, the overlap of identifications in replicate experiments is low (35-60%) (14,15).The limitations of the shotgun method have propelled a recent fervor in targeted proteomic methods-namely, selected reaction monitoring (SRM 1 , also known as MRM, multiple reaction monitoring) (16 -21). SRM achieves the reproducibility and sensitivity that the shotgun approach lacks, and even offers a route to determine absolute abundance (21).…”

mentioning

confidence: 99%

Parallel Reaction Monitoring for High Resolution and High Mass Accuracy Quantitative, Targeted Proteomics

Peterson

Russell

Bailey

et al. 2012

Molecular & Cellular Proteomics

1,107

953

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Selected reaction monitoring on a triple quadrupole mass spectrometer is currently experiencing a renaissance within the proteomics community for its, as yet, unparalleled ability to characterize and quantify a set of proteins reproducibly, completely, and with high sensitivity. Given the immense benefit that high resolution and accurate mass instruments have brought to the discovery proteomics field, we wondered if highly accurate mass measurement capabilities could be leveraged to provide benefits in the targeted proteomics domain as well. Here, we propose a new targeted proteomics paradigm centered on the use of next generation, quadrupole-equipped high resolution and accurate mass instruments: parallel reaction monitoring (PRM). In PRM, the third quadrupole of a triple quadrupole is substituted with a high resolution and accurate mass mass analyzer to permit the parallel detection of all target product ions in one, concerted high resolution mass analysis. We detail the analytical performance of the PRM method, using a quadrupoleequipped bench-top Orbitrap MS, and draw a performance comparison to selected reaction monitoring in terms of run-to-run reproducibility, dynamic range, and measurement accuracy. In addition to requiring minimal upfront method development and facilitating automated data analysis, PRM yielded quantitative data over a wider dynamic range than selected reaction monitoring in the presence of a yeast background matrix because of PRM's high selectivity in the mass-to-charge domain. With achievable linearity over the quantifiable dynamic range found to be statistically equal between the two methods, our investigation suggests that PRM will be a promising new addition to the quantitative proteomics toolbox. Molecular & Cellular Proteomics 11: 10.1074/ mcp.O112.020131, 1475-1488, 2012.The most widespread protein sequencing technique is the shotgun method. Proteins are digested into peptides, chromatographically separated, and measured by mass spectrometers (1-10). Many types of mass spectrometers are used-quadrupole ion traps, quadrupole ion trap hybrids such as the QLT (quadrupole linear ion trap)-Orbitrap or QLT-FT-ICR, and quadrupole time-of-flight (Q-TOF) hybrids-but, the experiments, from the MS measurement onward, are basically identical: the masses of eluting cationic peptide precursors are measured in a MS scan, and the most abundant precursors are selected in series for successive tandem MS events (MS/MS). This process, called data-dependent acquisition, continues for the duration of a chromatographic separation, and constant MS operation in this manner can generate hundreds of thousands of spectra in days. These spectra are then mapped to peptide or protein sequence databases using highly-evolved database search algorithms (11-13). Successful results can be obtained within just a few days and are nothing short of spectacular: tens of thousands of unique peptide spectral matches mapping to several thousand unique protein isoforms have become the norm. Although this approach certainly ...

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“…MS already has become the method of choice for quantifying protein levels, and a number of quantitative proteomics strategies for this purpose have been described (9)(10)(11)(12)(13)(14). Interestingly, MS already has been used to detect and precisely quantify somatic mutations at the DNA level but not at the protein level (15).…”

mentioning

confidence: 99%

Mutant proteins as cancer-specific biomarkers

Wang

Chaerkady

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

155

148

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Cancer biomarkers are currently the subject of intense research because of their potential utility for diagnosis, prognosis, and targeted therapy. In theory, the gene products resulting from somatic mutations are the ultimate protein biomarkers, being not simply associated with tumors but actually responsible for tumorigenesis. We show here that the altered protein products resulting from somatic mutations can be identified directly and quantified by mass spectrometry. The peptides expressed from normal and mutant alleles were detected by selected reaction monitoring (SRM) of their product ions using a triple-quadrupole mass spectrometer. As a prototypical example of this approach, we demonstrated that it is possible to quantify the number and fraction of mutant Ras protein present in cancer cell lines. There were an average of 1.3 million molecules of Ras protein per cell, and the ratio of mutant to normal Ras proteins ranged from 0.49 to 5.6. Similarly, we found that mutant Ras proteins could be detected and quantified in clinical specimens such as colorectal and pancreatic tumor tissues as well as in premalignant pancreatic cyst fluids. In addition to answering basic questions about the relative levels of genetically abnormal proteins in tumors, this approach could prove useful for diagnostic applications.genetics | proteomics | pancreatic cancer | genetic diagnosis | companion diagnostics

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Repeatability and Reproducibility in Proteomic Identifications by Liquid Chromatography−Tandem Mass Spectrometry

Cited by 509 publications

References 34 publications

Proteome profiling of extracellular vesicles captured with the affinity peptide Vn96: comparison of Laemmli and TRIzol© protein‐extraction methods

Proteome profiling of extracellular vesicles captured with the affinity peptide Vn96: comparison of Laemmli and TRIzol© protein‐extraction methods

Parallel Reaction Monitoring for High Resolution and High Mass Accuracy Quantitative, Targeted Proteomics

Mutant proteins as cancer-specific biomarkers

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