In-gel digestion for mass spectrometric characterization of proteins and proteomes
TL;DR: This protocol is for the in-gel digestion of both silver and Coomassie-stained protein spots or bands and can be followed by MALDI-MS or LC-MS/MS analysis to identify proteins at sensitivities better than a few femtomoles of protein starting material.
Abstract: In-gel digestion of proteins isolated by gel electrophoresis is a cornerstone of mass spectrometry (MS)-driven proteomics. The 10-year-old recipe by Shevchenko et al. has been optimized to increase the speed and sensitivity of analysis. The protocol is for the in-gel digestion of both silver and Coomassie-stained protein spots or bands and can be followed by MALDI-MS or LC-MS/MS analysis to identify proteins at sensitivities better than a few femtomoles of protein starting material.
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TL;DR: A proteomic-scale analysis of protein acetylation suggests that it is an important biological regulatory mechanism and the regulatory scope of lysine acetylations is broad and comparable with that of other major posttranslational modifications.
Abstract: Lysine acetylation is a reversible posttranslational modification of proteins and plays a key role in regulating gene expression. Technological limitations have so far prevented a global analysis of lysine acetylation's cellular roles. We used high-resolution mass spectrometry to identify 3600 lysine acetylation sites on 1750 proteins and quantified acetylation changes in response to the deacetylase inhibitors suberoylanilide hydroxamic acid and MS-275. Lysine acetylation preferentially targets large macromolecular complexes involved in diverse cellular processes, such as chromatin remodeling, cell cycle, splicing, nuclear transport, and actin nucleation. Acetylation impaired phosphorylation-dependent interactions of 14-3-3 and regulated the yeast cyclin-dependent kinase Cdc28. Our data demonstrate that the regulatory scope of lysine acetylation is broad and comparable with that of other major posttranslational modifications.
3,787 citations
Cites methods from "In-gel digestion for mass spectrome..."
...In-gel digestion of proteins and recovery of peptides were performed as described earlier (Shevchenko et al., 2006; Shevchenko et al., 1996)....
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Johns Hopkins University1, Johns Hopkins University School of Medicine2, University of Toronto3, University of La Frontera4, Imperial College London5, Post Graduate Institute of Medical Education and Research6, Armed Forces Medical College7, National Institute of Mental Health and Neurosciences8, Hong Kong University of Science and Technology9, University of Maryland, Baltimore10
TL;DR: A draft map of the human proteome is presented using high-resolution Fourier-transform mass spectrometry to discover a number of novel protein-coding regions, which includes translated pseudogenes, non-c coding RNAs and upstream open reading frames.
Abstract: The availability of human genome sequence has transformed biomedical research over the past decade. However, an equivalent map for the human proteome with direct measurements of proteins and peptides does not exist yet. Here we present a draft map of the human proteome using high-resolution Fourier-transform mass spectrometry. In-depth proteomic profiling of 30 histologically normal human samples, including 17 adult tissues, 7 fetal tissues and 6 purified primary haematopoietic cells, resulted in identification of proteins encoded by 17,294 genes accounting for approximately 84% of the total annotated protein-coding genes in humans. A unique and comprehensive strategy for proteogenomic analysis enabled us to discover a number of novel protein-coding regions, which includes translated pseudogenes, non-coding RNAs and upstream open reading frames. This large human proteome catalogue (available as an interactive web-based resource at http://www.humanproteomemap.org) will complement available human genome and transcriptome data to accelerate biomedical research in health and disease.
1,965 citations
TL;DR: It is shown that the receptor-binding region of pre-S1 specifically interacts with sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter predominantly expressed in the liver that is a functional receptor for HBV and HDV.
Abstract: Human hepatitis B virus (HBV) infection and HBV-related diseases remain a major public health problem. Individuals coinfected with its satellite hepatitis D virus (HDV) have more severe disease. Cellular entry of both viruses is mediated by HBV envelope proteins. The pre-S1 domain of the large envelope protein is a key determinant for receptor(s) binding. However, the identity of the receptor(s) is unknown. Here, by using near zero distance photo-cross-linking and tandem affinity purification, we revealed that the receptor-binding region of pre-S1 specifically interacts with sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter predominantly expressed in the liver. Silencing NTCP inhibited HBV and HDV infection, while exogenous NTCP expression rendered nonsusceptible hepatocarcinoma cells susceptible to these viral infections. Moreover, replacing amino acids 157-165 of nonfunctional monkey NTCP with the human counterpart conferred its ability in supporting both viral infections. Our results demonstrate that NTCP is a functional receptor for HBV and HDV.
1,662 citations
Cites methods from "In-gel digestion for mass spectrome..."
...Silver stained gel bands were cut, followed by in-gel reduction, alkylation, and trypsin digestion as previously described (Shevchenko et al., 2006) ....
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...Silver stained gel bands were cut, followed by in-gel reduction, alkylation, and trypsin digestion as previously described (Shevchenko et al., 2006)....
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TL;DR: High-resolution mass spectrometry–based proteomics was applied to investigate the proteome and phosphoproteome of the human cell cycle on a global scale and quantified 6027 proteins and 20,443 unique phosphorylation sites and their dynamics, finding that nuclear proteins and proteins involved in regulating metabolic processes have high phosphorylated site occupancy in mitosis, suggesting that these proteins may be inactivated by phosphorylate in mitotic cells.
Abstract: Eukaryotic cells replicate by a complex series of evolutionarily conserved events that are tightly regulated at defined stages of the cell division cycle. Progression through this cycle involves a large number of dedicated protein complexes and signaling pathways, and deregulation of this process is implicated in tumorigenesis. We applied high-resolution mass spectrometry-based proteomics to investigate the proteome and phosphoproteome of the human cell cycle on a global scale and quantified 6027 proteins and 20,443 unique phosphorylation sites and their dynamics. Co-regulated proteins and phosphorylation sites were grouped according to their cell cycle kinetics and compared to publicly available messenger RNA microarray data. Most detected phosphorylation sites and more than 20% of all quantified proteins showed substantial regulation, mainly in mitotic cells. Kinase-motif analysis revealed global activation during S phase of the DNA damage response network, which was mediated by phosphorylation by ATM or ATR or DNA-dependent protein kinases. We determined site-specific stoichiometry of more than 5000 sites and found that most of the up-regulated sites phosphorylated by cyclin-dependent kinase 1 (CDK1) or CDK2 were almost fully phosphorylated in mitotic cells. In particular, nuclear proteins and proteins involved in regulating metabolic processes have high phosphorylation site occupancy in mitosis. This suggests that these proteins may be inactivated by phosphorylation in mitotic cells.
1,447 citations
TL;DR: In-solution, online and on-column protocols for stable isotope dimethyl labeling of sample amounts ranging from sub-micrograms to milligrams are described, which uses inexpensive reagents and is applicable to virtually any sample.
Abstract: Accurate quantification of protein expression in biological systems is an increasingly important part of proteomics research. Incorporation of differential stable isotopes in samples for relative protein quantification has been widely used. Stable isotope incorporation at the peptide level using dimethyl labeling is a reliable, cost-effective and undemanding procedure that can be easily automated and applied in high-throughput proteomics experiments. Although alternative multiplex quantitative proteomics approaches introduce isotope labels at the organism level ('stable isotope labeling by amino acids in cell culture' (SILAC)) or enable the simultaneous analysis of eight samples (isobaric tagging for relative and absolute quantification (iTRAQ)), stable isotope dimethyl labeling is advantageous in that it uses inexpensive reagents and is applicable to virtually any sample. We describe in-solution, online and on-column protocols for stable isotope dimethyl labeling of sample amounts ranging from sub-micrograms to milligrams. The labeling steps take approximately 60–90 min, whereas the full protocol including digestion and (two-dimensional) liquid chromatography-mass spectrometry takes approximately 1.5–3 days to complete.
1,291 citations
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TL;DR: Silver staining allows a substantial shortening of sample preparation time and may, therefore, be preferable over Coomassie staining, and this work removes a major obstacle to the low-level sequence analysis of proteins separated on polyacrylamide gels.
Abstract: Proteins from silver-stained gels can be digested enzymatically and the resulting peptides analyzed and sequenced by mass spectrometry. Standard proteins yield the same peptide maps when extracted from Coomassie- and silver-stained gels, as judged by electrospray and MALDI mass spectrometry. The low nanogram range can be reached by the protocols described here, and the method is robust. A silver-stained one-dimensional gel of a fraction from yeast proteins was analyzed by nanoelectrospray tandem mass spectrometry. In the sequencing, more than 1000 amino acids were covered, resulting in no evidence of chemical modifications due to the silver staining procedure. Silver staining allows a substantial shortening of sample preparation time and may, therefore, be preferable over Coomassie staining. This work removes a major obstacle to the low-level sequence analysis of proteins separated on polyacrylamide gels.
8,437 citations
"In-gel digestion for mass spectrome..." refers methods in this paper
...) The in-gel digestion protocol presented here was originally introduced in 1996 (ref...
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TL;DR: The ability of mass spectrometry to identify and, increasingly, to precisely quantify thousands of proteins from complex samples can be expected to impact broadly on biology and medicine.
Abstract: Recent successes illustrate the role of mass spectrometry-based proteomics as an indispensable tool for molecular and cellular biology and for the emerging field of systems biology. These include the study of protein-protein interactions via affinity-based isolations on a small and proteome-wide scale, the mapping of numerous organelles, the concurrent description of the malaria parasite genome and proteome, and the generation of quantitative protein profiles from diverse species. The ability of mass spectrometry to identify and, increasingly, to precisely quantify thousands of proteins from complex samples can be expected to impact broadly on biology and medicine.
6,597 citations
"In-gel digestion for mass spectrome..." refers background in this paper
...468" language="eng" publish="issue" relation="no"> Introduction Many proteomics efforts rely on the pre-separation of target proteins by one- or two-dimensional gel electrophoresi...
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TL;DR: SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system and is applied to the relative quantitation of changes in protein expression during the process of muscle cell differentiation.
5,653 citations
"In-gel digestion for mass spectrome..." refers methods in this paper
...or by mixing SILAC-labeled protein mixtures before separatio...
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TL;DR: A novel procedure in which a very small disk of beads embedded in a Teflon meshwork is placed as a microcolumn into pipet tips, finding that the Stage system is well-suited as a universal sample preparation system for proteomics.
Abstract: Proteomics is critically dependent on optimal sample preparation. Particularly, the interface between protein digestion and mass spectrometric analysis has a large influence on the overall quality and sensitivity of the analysis. We here describe a novel procedure in which a very small disk of beads embedded in a Teflon meshwork is placed as a microcolumn into pipet tips. Termed Stage, for STop And Go Extraction, the procedure has been implemented with commercially available material (C18 Empore Disks (3M, Minneapolis, MN)) as frit and separation material. The disk is introduced in a simple and fast process yielding a convenient and completely reliable procedure for the production of self-packed microcolumns in pipet tips. It is held in place free of obstacles solely by the narrowing tip, ensuring optimized loading and elution of analytes. Five disks are conveniently placed in 1 min, adding 300 μL/...
2,358 citations
"In-gel digestion for mass spectrome..." refers methods in this paper
...Peptide mixtures can also be purified before LC MS/MS on STAGE (Stop and go extraction) tips before loadin...
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TL;DR: Two recently developed methodologies offer the opportunity to obtain quantitative proteomic information by comparing the signals from the same peptide under different conditions, and stable isotope labels facilitates direct quantification from the mass spectra.
Abstract: The field of proteomics is built on technologies to analyze large numbers of proteins--ideally the entire proteome--in the same experiment. Mass spectrometry (MS) has been successfully used to characterize proteins in complex mixtures, but results so far have largely been qualitative. Two recently developed methodologies offer the opportunity to obtain quantitative proteomic information. Comparing the signals from the same peptide under different conditions yields a rough estimate of relative protein abundance between two proteomes. Alternatively, and more accurately, peptides are labeled with stable isotopes, introducing a predictable mass difference between peptides from two experimental conditions. Stable isotope labels can be incorporated 'post-harvest', by chemical approaches or in live cells through metabolic incorporation. This isotopic handle facilitates direct quantification from the mass spectra. Using these quantitative approaches, precise functional information as well as temporal changes in the proteome can be captured by MS.
1,621 citations
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...Furthermore, combined with quantitative proteomics method...
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