Identification of 2D-gel proteins: A comparison of MALDI/TOF peptide mass mapping to <i>μ</i> LC-ESI tandem mass spectrometry

Lim, H.; Eng, Jimmy K.; Yates, John R.; Tollaksen, Sandra L.; Giometti, Carol S.; Holden, James F.; Adams, Michael W. W.; Reich, Claudia I.; Olsen, Gary J.; Hays, Lara G.

doi:10.1016/s1044-0305(03)00144-2

Cited by 124 publications

(101 citation statements)

References 51 publications

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“…This quantitative approach is based on the assumptions that the protein spots can be well resolved and that the changes in the normalized spot image intensity can be correlated with the expression level of particular proteins. However, as reported previously [16] and described here, multiple proteins are commonly identified from apparently single, well-resolved gel spots. Lim et al [16] reported that up to 60% of 2-D gel spots analyzed by LC-MS/MS contained multiple proteins per spot.…”

Section: Resultssupporting

confidence: 80%

“…However, as reported previously [16] and described here, multiple proteins are commonly identified from apparently single, well-resolved gel spots. Lim et al [16] reported that up to 60% of 2-D gel spots analyzed by LC-MS/MS contained multiple proteins per spot. Even with a relatively narrower pH range of 4-7 for IEF, more than 20% of the analyzed spots have been found to contain multiple proteins [32].…”

Section: Resultssupporting

confidence: 80%

“…By implementing an LC separation, it is possible to minimize ion suppression and achieve peptide enrichment to improve detection sensitivity and dynamic range. In particular, on-line capillary (Cap) or nano-LC interfaced with a tandem mass spectrometer operated in data-dependent MS/MS acquisition mode will make the second stage analysis more comprehensive and allows higher throughput [16].…”

Section: Introductionmentioning

confidence: 99%

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Development of an integrated approach for evaluation of 2‐D gel image analysis: Impact of multiple proteins in single spots on comparative proteomics in conventional 2‐D gel/MALDI workflow

Yang¹,

Thannhauser²,

Li³

et al. 2007

Electrophoresis

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With 2-D gel mapping, it is often observed that essentially identical proteins migrate to different positions in the gel, while some seemingly well-resolved protein spots consist of multiple proteins. These observations can undermine the validity of gel-based comparative proteomic studies. Through a comparison of protein identifications using direct MALDI-TOF/ TOF and LC-ESI-MS/MS analyses of 2-D gel separated proteins from cauliflower florets, we have developed an integrated approach to improve the accuracy and reliability of comparative 2-D electrophoresis. From 46 spots of interest, we identified 51 proteins by MALDI-TOF/TOF analysis and 108 proteins by LC-ESI-MS/MS. The results indicate that 75% of the analyzed spots contained multiple proteins. A comparison of hit rank for protein identifications showed that 37 out of 43 spots identified by MALDI matched the top-ranked hit from the ESI-MS/MS. By using the exponentially modified protein abundance index (emPAI) to determine the abundance of the individual component proteins for the spots containing multiple proteins, we found that the top-hit proteins from 40 out of 43 spots identified by MALDI matched the most abundant proteins determined by LC-MS/MS. Furthermore, our 2-D-GeLC-MS/MS results show that the top-hit proteins in 44 identified spots contributed on average 81% of the spots' staining intensity. This is the first quantitative measurement of the average rate of false assignment for direct MALDI analysis of 2-D gel spots using a new integrated workflow (2-D gel imaging, "2-D GeLC-MS/MS", and emPAI analysis). Here, the new approach is proposed as an alternative to traditional gel-based quantitative proteomics studies.

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Section: Resultssupporting

confidence: 80%

Section: Resultssupporting

confidence: 80%

Section: Introductionmentioning

confidence: 99%

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Development of an integrated approach for evaluation of 2‐D gel image analysis: Impact of multiple proteins in single spots on comparative proteomics in conventional 2‐D gel/MALDI workflow

Yang¹,

Thannhauser²,

Li³

et al. 2007

Electrophoresis

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“…It provides high sensitivity and reliability, and allows to reproducibly and to effectively match data to the predicted proteins (Lim et al 2003). The possibility of using LC-MS could be alternative to obtained better results.…”

Section: Comparison Of Maldi-tof With Other Methodsmentioning

confidence: 99%

Proteomic signature of fenugreek treated by methyl jasmonate and cholesterol

et al. 2017

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Main conclusion Changes in proteome level as a result of methyl jasmonate and cholesterol treatment were investigated. The identified proteins were often involved in response to stress caused by various treatments. Furthermore, 18 proteins were expressed in treatmentspecific manner. In this study, the fenugreek plants were treated with methyl jasmonate (as an elicitor) and cholesterol (as a precursor of steroids and steroidal saponins) to check reaction at the level of the proteome to stress and to investigate steroidal saponin (diosgenin) biosynthesis. Proteins were separated by two-dimensional electrophoresis (2-DE) and identified by MALDI-ToF/ToF followed by database searches using Mascot search engine. Totally, 63 and 41 protein spots were differentially expressed after methyl jasmonate and cholesterol treatment, respectively. These proteins were classified into seven groups: photosynthesis, energy, metabolism, protein metabolism, secondary metabolism, stress and defense, and other. We found that 9 proteins were responsive to all treatments, and 18 proteins expressed in treatment-specific manner. Higher level of photosynthetic proteins sensitive to both biotic and abiotic stimuli was detected. In addition, proteins related to the stress (especially oxidative) and defense, protein, and secondary metabolism were overexpressed. The results indicate that methyl jasmonate and cholesterol elicited a defense reaction at the proteome level as a response to stress. The usefulness of 2-DE method for identification of proteins related with species-specific metabolic pathways is restricted. Integration of transcriptome data with proteomic analysis improved annotation process.

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“…The second approach is to modify the commercially available ZipTips from Millipore with a conductive electrode so they can be used directly on the Nanomate high-throughput platform. Much like the Poros resins, ZipTips have shown their utility for desalting and concentrating peptide, as well as protein samples [10,14,15]. The final approach is to modify the Nanomate itself such that a solvent gradient can be delivered from a liquid chromatography system through the Nanomate and across a disposable, either self-packed or commercial, chromatographic pipette tip.…”

mentioning

confidence: 99%

Disposable chromatography for a high-throughput nano-ESI/MS and nano-ESI/MS-MS platform

Williams

Tomer

2004

J. Am. Soc. Mass Spectrom.

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High-throughput proteomics has typically relied on protein identification based on MALDI-MS peptide maps of proteolytic digests of 2D-gel-separated proteins. This technique, however, requires significant sequence coverage in order to achieve a high level of confidence in the identification. Tandem MS data have the advantage of requiring fewer peptides (2) for high confidence identification, assuming adequate MS/MS sequence coverage. MALDI-MS/MS techniques are becoming available, but can still be problematic because of the difficulty of inducing fragment ions of a singly charged parent ion. Electrospray ionization, however, has the advantage of generating multiply charged species that are more readily fragmented during MS/MS analysis. Two electrospray/tandem mass spectrometry-based approaches, nanovial-ESI-MS/MS and LC-MS/MS, are used for high throughput proteomics, but much less often than MALDI-MS and peptide mass fingerprinting. Nanovial introduction entails extensive manual manipulation and often shows significant chemical background from the in-gel digest. LC-MS has the advantages that the chemical background can be removed prior to analysis and the analytes are concentrated during the separation, resulting in more abundant analyte signals. On the other hand, LC-MS can often be time intensive. Here, we report the incorporation of on-line sample clean-up and analyte concentration with a high-throughput, chip-based, robotic nano-ESI-MS platform for proteomics studies. is well suited for protein identification from gel band digests because of the relative ease and availability of modern ESI-MS/MS techniques. Detection of far fewer peptides is necessary for unambiguous identification when tandem MS methods are incorporated than when only MS techniques are employed [7]. ESI-MS/MS has an advantage over matrix assisted laser desorption ionization (MALDI)-MS/MS [8] in that multiply charged species, which are more readily fragmented in tandem MS applications than singly charged species at equivalent collision energies, are formed during the electrospray process. Moreover, many labs are already equipped with mass spectrometers that are capable of ESI-MS/MS whereas MALDI instruments with tandem mass analyzers have become commercially available only in the last few years. Drawbacks of using ESI-MS or ESI-MS/MS as the final analytical step for protein identification, however, are the high chemical background and low sample abundance of proteolytic digests of gel bands. These problems can be overcome with on-line liquid chromatography (LC) or sample clean-up and concentration prior to ESI-MS, but neither of these techniques is conducive to high-throughput applications.In an effort to achieve high-throughput clean-up for ESI-MS and ESI-MS/MS analysis of peptides from gel band tryptic digests, we have adapted several approaches for concentrating and desalting these tryptic peptides for implementation with a robotic nanospray system, the Advion Biosciences Nanomate 100 [9 -11].

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Identification of 2D-gel proteins: A comparison of MALDI/TOF peptide mass mapping to μ LC-ESI tandem mass spectrometry

Cited by 124 publications

References 51 publications

Development of an integrated approach for evaluation of 2‐D gel image analysis: Impact of multiple proteins in single spots on comparative proteomics in conventional 2‐D gel/MALDI workflow

Development of an integrated approach for evaluation of 2‐D gel image analysis: Impact of multiple proteins in single spots on comparative proteomics in conventional 2‐D gel/MALDI workflow

Proteomic signature of fenugreek treated by methyl jasmonate and cholesterol

Disposable chromatography for a high-throughput nano-ESI/MS and nano-ESI/MS-MS platform

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