Limitations and Pitfalls in Protein Identification by Mass Spectrometry

Lubec, Gert; Afjehi‐Sadat, Leila

doi:10.1021/cr068213f

Cited by 111 publications

(45 citation statements)

References 288 publications

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“…SDS-PAGE in combination with LC-MS/MS is generally limited to the analysis of proteins above 10 kDa, and a low molecular weight is a known limitation in protein identification by MS [36], [37]. Since the majority of the predicted alternative proteome is composed of proteins less than 90 amino acids long which have a predicted molecular weight below 10 kDa (Fig.…”

Section: Resultsmentioning

confidence: 99%

Direct Detection of Alternative Open Reading Frames Translation Products in Human Significantly Expands the Proteome

et al. 2013

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A fully mature mRNA is usually associated to a reference open reading frame encoding a single protein. Yet, mature mRNAs contain unconventional alternative open reading frames (AltORFs) located in untranslated regions (UTRs) or overlapping the reference ORFs (RefORFs) in non-canonical +2 and +3 reading frames. Although recent ribosome profiling and footprinting approaches have suggested the significant use of unconventional translation initiation sites in mammals, direct evidence of large-scale alternative protein expression at the proteome level is still lacking. To determine the contribution of alternative proteins to the human proteome, we generated a database of predicted human AltORFs revealing a new proteome mainly composed of small proteins with a median length of 57 amino acids, compared to 344 amino acids for the reference proteome. We experimentally detected a total of 1,259 alternative proteins by mass spectrometry analyses of human cell lines, tissues and fluids. In plasma and serum, alternative proteins represent up to 55% of the proteome and may be a potential unsuspected new source for biomarkers. We observed constitutive co-expression of RefORFs and AltORFs from endogenous genes and from transfected cDNAs, including tumor suppressor p53, and provide evidence that out-of-frame clones representing AltORFs are mistakenly rejected as false positive in cDNAs screening assays. Functional importance of alternative proteins is strongly supported by significant evolutionary conservation in vertebrates, invertebrates, and yeast. Our results imply that coding of multiple proteins in a single gene by the use of AltORFs may be a common feature in eukaryotes, and confirm that translation of unconventional ORFs generates an as yet unexplored proteome.

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Section: Resultsmentioning

confidence: 99%

Direct Detection of Alternative Open Reading Frames Translation Products in Human Significantly Expands the Proteome

et al. 2013

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“…These may arise from protease impurities, contamination with other proteases and nonspecific proteolysis during sample preparation. 65 In addition, incubation time, digestion buffers, digestion temperature, 65 and enzyme to protein ratio, 66 have also been shown to affect enzyme specificity. Recently, the specificity of Glu-C, chymotrypsin and CNBr digestion was assessed by Pevzner and coworkers using Shewanella oneidensis as the model system and the frequency of unspecific cleavages was reported and evaluated.…”

Section: Sample Preparation Liquid Chromatocraphy and Bioinformaticsmentioning

confidence: 99%

Evaluation and Optimization of Mass Spectrometric Settings during Data-dependent Acquisition Mode: Focus on LTQ-Orbitrap Mass Analyzers

et al. 2013

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Mass-spectrometry-based proteomics has evolved as the preferred method for the analysis of complex proteomes. Undoubtedly recent advances in mass spectrometry instrumentation have greatly enhanced proteomic analysis. A popular instrument platform in proteomics research is the LTQ-Orbitrap mass analyzer. In this tutorial we discuss the significance of evaluating and optimizing mass spectrometric settings on the LTQ-Orbitrap during CID data-dependent acquisition (DDA) mode to improve protein and peptide identification rates. We focus on those MS and MS/MS parameters, which have been systematically examined and evaluated by several researchers and are commonly used during DDA. More specifically we discuss the effect of mass resolving power, preview mode for FTMS scan, monoisotopic precursor selection, signal threshold for triggering MS/MS events, number of microscans per MS/MS scan, number of MS/MS events, automatic gain control target value (ion population) for MS and MS/MS, maximum ion injection time for MS/MS, rapid and normal scan rate and prediction of ion injection time. We, furthermore, present data from the latest generation LTQ-Orbitrap system, the Orbitrap Elite, along with recommended MS and MS/MS parameters. The Orbitrap Elite outperforms the Orbitrap Classic in terms of scan speed, sensitivity, dynamic range, resolving power and resulting in higher identification rates. Several of the optimized MS parameters determined on the LTQ-Orbitrap Classic and XL were easily transferable to the Orbitrap Elite, whereas others needed to be reevaluated. Finally, the Q Exactive and HCD are briefly discussed, as well as, sample preparation, LC-optimization and bioinformatics analysis. We hope this tutorial will serve as guidance for researchers new to the field of proteomics and assist in achieving optimal results.

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“…For example, a common approach is to purify a protein complex from crude cell lysates and then identify its components by mass spectrometry (Aeber-sold and Mann, 2003; Nilsson et al, 2010). However, many well-acknowledged technical limitations diminish the ability to identify important interacting proteins that are only present at a few copies per cell or that interact with their binding partner only transiently (Hamdan and Righetti, 2002; Hein et al, 2015; Lubec and Afjehi-Sadat, 2007). An approach that partly circumvents these limitations is the yeast 2-hybrid (Y2H) assay, which exploits engineered yeast cells that grow only when a protein of interest binds to a fragment of an interacting partner (Fields and Song, 1989).…”

Section: Introductionmentioning

confidence: 99%

DEEPN as an Approach for Batch Processing of Yeast 2-Hybrid Interactions

et al. 2016

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Summary We adapted the yeast 2-hybrid assay to simultaneously uncover multiple transient protein interactions within a single screen by using a strategy termed DEEPN (dynamic enrichment for evaluation of protein networks). This approach incorporates high-throughput DNA sequencing and computation to follow competition among a plasmid population encoding interacting partners. To demonstrate the capacity of DEEPN, we identify a wide range of ubiq-uitin-binding proteins, including interactors that we verify biochemically. To demonstrate the specificity of DEEPN, we show that DEEPN allows simultaneous comparison of candidate interactors across multiple bait proteins, allowing differential interactions to be identified. This feature was used to identify interactors that distinguish between GTP- and GDP-bound conformations of Rab5.

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Limitations and Pitfalls in Protein Identification by Mass Spectrometry

Cited by 111 publications

References 288 publications

Direct Detection of Alternative Open Reading Frames Translation Products in Human Significantly Expands the Proteome

Direct Detection of Alternative Open Reading Frames Translation Products in Human Significantly Expands the Proteome

Evaluation and Optimization of Mass Spectrometric Settings during Data-dependent Acquisition Mode: Focus on LTQ-Orbitrap Mass Analyzers

DEEPN as an Approach for Batch Processing of Yeast 2-Hybrid Interactions

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