Stop and Go Extraction Tips for Matrix-Assisted Laser Desorption/Ionization, Nanoelectrospray, and LC/MS Sample Pretreatment in Proteomics

Rappsilber, Juri; Ishihama, Yasushi; Mann, Matthias

doi:10.1021/ac026117i

Cited by 2,464 publications

(2,106 citation statements)

References 24 publications

Supporting

Mentioning

2,094

Contrasting

Unclassified

Order By: Relevance

“…Peptides were eluted from the beads twice using 100 l 0.1% TFA, and fractions were then combined and lyophilized. Eluted peptides were desalted prior to LC-MS/MS analysis using in-house C18 STAGE tips as previously described (37).…”

Section: Silacmentioning

confidence: 99%

Quantitative Profiling of the Activity of Protein Lysine Methyltransferase SMYD2 Using SILAC-Based Proteomics

Olsen

Cao

Han

et al. 2016

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

The significance of non-histone lysine methylation in cell biology and human disease is an emerging area of research exploration. The development of small molecule inhibitors that selectively and potently target enzymes that catalyze the addition of methyl-groups to lysine residues, such as the protein lysine mono-methyltransferase SMYD2, is an active area of drug discovery. Critical to the accurate assessment of biological function is the ability to identify target enzyme substrates and to define enzyme substrate specificity within the context of the cell. Here, using stable isotopic labeling with amino acids in cell culture (SILAC) coupled with immunoaffinity enrichment of mono-methyl-lysine (Kme1) peptides and mass spectrometry, we report a comprehensive, large-scale proteomic study of lysine mono-methylation, comprising a total of 1032 Kme1 sites in esophageal squamous cell carcinoma (ESCC) cells and 1861 Kme1 sites in ESCC cells overexpressing SMYD2. Among these Kme1 sites is a subset of 35 found to be potently down-regulated by both shRNA-mediated knockdown of SMYD2 and LLY-507, a selective small molecule inhibitor of SMYD2. In addition, we report specific protein sequence motifs enriched in Kme1 sites that are directly regulated by endogenous SMYD2 activity, revealing that SMYD2 substrate specificity is more diverse than expected. We further show direct activity of SMYD2 toward BTF3-K2, PDAP1-K126 as well as numerous sites within the repetitive units of two unique and exceptionally large proteins, AHNAK and AHNAK2. Collectively, our findings provide quantitative insights into the cellular activity and substrate recognition of SMYD2 as well as the global landscape and regulation of protein mono-methylation. Protein lysine methyltransferases (PKMTs) 1 catalyze the sequence-specific transfer of one, two, or three methyl groups to the side chains of lysine residues (1-4). In addition to the extensively studied lysine methylation on histones, PKMTs can modify non-histone proteins (3,(5)(6)(7)(8)). An increasing number of non-histone proteins have been reported as PKMT substrates, and, as a result, potential roles for the dysregulation of non-histone lysine methylation in cancer development and progression have been proposed (9). The majority of PKMT substrates have been identified based on biochemical methylation assays using recombinant enzyme and substrate, followed by cell-based assays typically requiring overexpression of enzyme and/or substrate to maximize signal detection (10 -13). However, it is difficult to discern whether these substrates are bona fide physiological substrates of endogenous PKMT activity. With the development of PKMT-targeted drugs emerging as a key area of drug discovery (14 -18), unbiased and quantitative methods enabling the comprehensive identification of histone and non-histone substrates in cells are critical to clarifying the cell-relevant substrates of these enzymes and to more accurately understand the functions of non-histone methylation.Progressing from targeted biochemical...

show abstract

Section: Silacmentioning

confidence: 99%

Quantitative Profiling of the Activity of Protein Lysine Methyltransferase SMYD2 Using SILAC-Based Proteomics

Olsen

Cao

Han

et al. 2016

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

show abstract

“…1A) were excised; proteins were reduced and alkylated with 10 mM DTT and 55 mM iodoacetamide, as described (32), and digested with 12.5 ng/ml trypsin (proteomics grade; Sigma). The resulting peptides were acidified to 1% trifluoroacetic acid (TFA; reagent grade; Sigma) and loaded onto Stage Tips (33). Peptides bound to the solid phase were washed with 50 ml 1% TFA and then eluted with 50 ml buffer B (80% acetonitrile, 0.1% TFA) and lyophilized in a SpeedVac Concentrator.…”

Section: Mass Spectrometrymentioning

confidence: 99%

Kidins220/ARMS Associates with B-Raf and the TCR, Promoting Sustained Erk Signaling in T Cells

Deswal

Meyer

Fiala

et al. 2013

The Journal of Immunology

View full text Add to dashboard Cite

The activation kinetics of MAPK Erk are critical for T cell development and activation. In particular, sustained Erk signaling is required for T cell activation and effector functions, such as IL-2 production. Although Raf-1 triggers transient Erk activation, B-Raf is implicated in sustained Erk signaling after TCR stimulation. In this study, we show that B-Raf is dephosphorylated on its inhibitory serine 365 upon TCR triggering. However, it is unknown how B-Raf activation is coupled to the TCR. Using mass spectrometry, we identified protein kinase D–interacting substrate of 220 kDa (Kidins220)/ankyrin repeat-rich membrane spanning protein, mammalian target of rapamycin, Rictor, Dock2, and GM130 as novel B-Raf interaction partners. We focused on Kidins220, a protein that has been studied in neuronal cells and found that it associated with the pre-TCR, αβTCR, and γδTCR. Upon prolonged TCR stimulation, the Kidins220–TCR interaction was reduced, as demonstrated by immunoprecipitation and proximity ligation assays. We show that Kidins220 is required for TCR-induced sustained, but not transient, Erk activation. Consequently, induction of the immediate early gene products and transcription factors c-Fos and Erg-1 was blocked, and upregulation of the activation markers CD69, IL-2, and IFN-γ was reduced. Further, Kidins220 was required for optimal calcium signaling. In conclusion, we describe Kidins220 as a novel TCR-interacting protein that couples B-Raf to the TCR. Kidins220 is mandatory for sustained Erk signaling; thus, it is crucial for TCR-mediated T cell activation.

show abstract

“…Solubilized gel slices were treated with dithiothreitol (DTT) and 55 mM iodoacetamide prior to in-gel digestion with sequencing grade trypsin at a 20:1 protein:enzyme ratio for 12 h at room temperature [66]. All peptide digests were desalted using homemade C18 STAGE tips [67] and diluted in 10 µl of 0.1% acetic acid for MS analysis. For quantitative (SILAC-based) mass spectrometry preparation, samples were treated with DTT and iodoacetamide and subjected to in-solution trypsin digest overnight prior to desalting and subsequent SCX fractionation as described previously [68].…”

Section: Sample Preparation and Mass Spectrometrymentioning

confidence: 99%

Global secretome analysis identifies novel mediators of bone metastasis

et al. 2012

View full text Add to dashboard Cite

Bone is the one of the most common sites of distant metastasis of solid tumors. Secreted proteins are known to influence pathological interactions between metastatic cancer cells and the bone stroma. To comprehensively profile secreted proteins associated with bone metastasis, we used quantitative and non-quantitative mass spectrometry to globally analyze the secretomes of nine cell lines of varying bone metastatic ability from multiple species and cancer types. By comparing the secretomes of parental cells and their bone metastatic derivatives, we identified the secreted proteins that were uniquely associated with bone metastasis in these cell lines. We then incorporated bioinformatic analyses of large clinical metastasis datasets to obtain a list of candidate novel bone metastasis proteins of several functional classes that were strongly associated with both clinical and experimental bone metastasis. Functional validation of selected proteins indicated that in vivo bone metastasis can be promoted by high expression of (1) the salivary cystatins CST1, CST2, and CST4; (2) the plasminogen activators PLAT and PLAU; or (3) the collagen functionality proteins PLOD2 and COL6A1. Overall, our study has uncovered several new secreted mediators of bone metastasis and therefore demonstrated that secretome analysis is a powerful method for identification of novel biomarkers and candidate therapeutic targets.

show abstract

Stop and Go Extraction Tips for Matrix-Assisted Laser Desorption/Ionization, Nanoelectrospray, and LC/MS Sample Pretreatment in Proteomics

Cited by 2,464 publications

References 24 publications

Quantitative Profiling of the Activity of Protein Lysine Methyltransferase SMYD2 Using SILAC-Based Proteomics

Quantitative Profiling of the Activity of Protein Lysine Methyltransferase SMYD2 Using SILAC-Based Proteomics

Kidins220/ARMS Associates with B-Raf and the TCR, Promoting Sustained Erk Signaling in T Cells

Global secretome analysis identifies novel mediators of bone metastasis

Contact Info

Product

Resources

About