Status of complete proteome analysis by mass spectrometry: SILAC labeled yeast as a model system

Godoy, Lyris Martins Franco de; Olsen, Jesper V.; Souza, Gustavo; Li, Guoqing; Mortensen, Poul Erik; Mann, Matthias

doi:10.1186/gb-2006-7-6-r50

Cited by 256 publications

(86 citation statements)

References 43 publications

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“…More importantly, the relative abundance of the bound factors will change only slightly or not at all. Because our LC tandem MS proteomic platform using the LTQ-FT is not so much limited by sample amount as by sample complexity, we do not expect any negative effect on our analysis (de Godoy et al 2006). Doublelabeling of the extracts with heavy lysine and arginine derivatives will roughly double the number of peptides that can be quantified and hence increase sensitivity.…”

Section: Discussionmentioning

confidence: 99%

A SILAC-based DNA protein interaction screen that identifies candidate binding proteins to functional DNA elements

2008

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Determining the underlying logic that governs the networks of gene expression in higher eukaryotes is an important task in the post-genome era. Sequence-specific transcription factors (TFs) that can read the genetic regulatory information and proteins that interpret the information provided by CpG methylation are crucial components of the system that controls the transcription of protein-coding genes by RNA polymerase II. We have previously described Stable Isotope Labeling by Amino acids in Cell culture (SILAC) for the quantitative comparison of proteomes and the determination of proteinprotein interactions. Here, we report a generic and scalable strategy to uncover such DNA protein interactions by SILAC that uses a fast and simple one-step affinity capture of TFs from crude nuclear extracts. Employing mutated or nonmethylated control oligonucleotides, specific TFs binding to their wild-type or methyl-CpG bait are distinguished from the vast excess of copurifying background proteins by their peptide isotope ratios that are determined by mass spectrometry. Our proof of principle screen identifies several proteins that have not been previously reported to be present on the fully methylated CpG island upstream of the human metastasis associated 1 family, member 2 gene promoter. The approach is robust, sensitive, and specific and offers the potential for high-throughput determination of TF binding profiles.

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Section: Discussionmentioning

confidence: 99%

A SILAC-based DNA protein interaction screen that identifies candidate binding proteins to functional DNA elements

2008

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“…These studies highlighted an unexpected diversity of mRNA species, raising the question of their functional relevance. Whereas it has been demonstrated by a proteomic approach that protein synthesis is slightly active in the sperm mitochondria (Zhao et al 2009), some of these mRNAs could also be delivered to the ovocyte and play further roles in early embryogenesis (Dadoune 2009). From another aspect, evaluating the spermatozoa RNA content using DNA microarrays provided meaningful results to assess sperm quality and/or to elucidate some cases of male sterility (Platts et al 2007;Garrido et al 2009).…”

Section: (D) Expression Profiling Of the Embryonic Germ Cellsmentioning

confidence: 99%

“…Secondarily, the problem of resolving the wide dynamic range in protein expression remains since there is no technology available to amplify low-copy number proteins in a biological sample. Nevertheless, instrument sensitivity is rapidly improving and, although still incomplete, extensive proteome coverage can now be achieved in large proteome experiments (de Godoy et al 2006(de Godoy et al , 2008. Likewise, absolute quantification of proteins in complex mixtures is becoming accessible thanks to innovative strategies such as the protein standard absolute quantification (PSAQ) technique (Brun et al 2007).…”

Section: Conclusion and Future Directions: Not All That Glitters Is Goldmentioning

confidence: 99%

Testicular postgenomics: targeting the regulation of spermatogenesis

Rolland

Jégou

Pineau

2010

Phil. Trans. R. Soc. B

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Sperm are, arguably, the most differentiated cells produced within the body of any given species. This is owing to the fact that spermatogenesis is an intricate and highly specialized process evolved to suit the individual particularities of each sexual species. Despite a vast diversity in method, the aim of spermatogenesis is always the same, the idealized transmission of genetic patrimony. Towards this goal certain requirements must always be met, such as a relative twofold reduction in ploidy, repackaging of the chromatin for transport and specialized enhancements for cell motility, recognition and fusion. In the past 20 years, the study of molecular networks coordinating male germ cell development, particularly in mammals, has become more and more facilitated thanks to large-scale analyses of genome expression. Such postgenomic endeavors have generated landscapes of data for both fundamental and clinical reproductive biology. Continuous, large-scale integration analyses of these datasets are undertaken which provide access to very precise information on a myriad of biomolecules. This review presents commonly used transcriptomic and proteomic workflows applied to various testicular germ cell studies. We will also provide a general overview of the technical possibilities available to reproductive genomic biologists, noting the advantages and drawbacks of each technique.

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“…A variety of first dimensions, not all of them LC-based, have been used, including size-exclusion chromatography (SEC), strong-cation exchange (SCX), strong-anion exchange, SDS-PAGE, and isoelectric focusing (IEF) techniques (e.g., immobilized pH gradient IEF and capillary IEF). 3,9,10,14,15,[18][19][20][21][22] Several factors are important for the first dimensionsit should have a large loading capacity, be configurable with the second dimension, and have solvent compatibility with the second mode. Several of the first-dimension methods fit these criteria, whereas others are better suited for off-line applications.…”

Section: Standard Combinations In Shotgun Strategiesmentioning

confidence: 99%

Multidimensional LC Separations in Shotgun Proteomics

2008

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Two modes of separation coupled with MS enable researchers to study complicated biological structures.Proteins are the molecular products of genes and play a central role in many biological processes. Protein expression occurs as a function of cellular and environmental conditions, and consequently proteins are expressed at different times and under different conditions. Insight is thus garnered by determining how protein expression has changed in a biological context.Over the past two decades, MS has become an important tool for the analysis of proteins. 1,2 One current method for the analysis of protein mixtures is proteolytic digestion followed by LC/MS/ MS. Once hard-to-handle proteins are converted into chemically well-behaved peptides, the approach overcomes many difficulties associated with protein mixture analysis. 3 Tandem MS has been particularly effective because the data can be directly used to identify peptides and subsequently infer which proteins (digested or undigested) are in the mixture. 4 This type of approach for the analysis of protein mixtures is often referred to as "shotgun" proteomics ( Figure 1).Because increasingly complicated biological structures are studied by tandem MS, the need for more powerful and highly resolving separation methods has grown. Consequently, the development and use of multidimensional LC (MDLC) in proteomics has thrived over the past few years. MDLC combines two or more forms of LC to increase the peak capacity, and thus the resolving power, of separations to better fractionate peptides prior to entering the mass spectrometer.High-resolution separation serves two functions in combination with MS. It better resolves peptides differing in charge and hydrophobicity to minimize ion suppression and improve ionization efficiency, and it simplifies the complexity of peptide ions entering the mass spectrometer to minimize undersampling. This last aspect is important because the tandem MS process is driven by data-dependent data acquisition and has a finite cycle time. A higher peak capacity and better resolving power improve the acquisition of data and can lead to a better representation of the proteins in the mixture. Improved resolution also results in a larger dynamic range.Although MDLC is by no means a new concept and has a long history, it has been enjoying a renaissance in proteomics. 5 In this article, we will provide an overview of the background, important applications, and future prospects for MDLC; in particular, we will focus on applications involving peptides. (To listen to a podcast about this feature, please go to the Analytical Chemistry website at pubs.acs.org/ac.)

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Status of complete proteome analysis by mass spectrometry: SILAC labeled yeast as a model system

Cited by 256 publications

References 43 publications

A SILAC-based DNA protein interaction screen that identifies candidate binding proteins to functional DNA elements

A SILAC-based DNA protein interaction screen that identifies candidate binding proteins to functional DNA elements

Testicular postgenomics: targeting the regulation of spermatogenesis

Multidimensional LC Separations in Shotgun Proteomics

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