Five-plex isotope dimethyl labeling for quantitative proteomics

Wu, Yue; Wang, Fangjun; Liu, Zheyi; Qin, Hongqiang; Song, Chunxia; Huang, Junfeng; Bian, Yangyang; Wei, Xiaoluan; Dong, Jing; Zou, Hanfa

doi:10.1039/c3cc47998f

Cited by 52 publications

(49 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…By using other reduction conditions, specific types of cysteine modifications (e.g., nitrosylation, glutathionylation) can be analyzed with OxcysDML [23]. In the dimethylation step, only two sample channels are labeled, which could be expanded to incorporate more samples by using sodium cyanborohydride and formaldehyde containing different heavy atoms [34] or isobaric tagging methods [23].…”

Section: Resultsmentioning

confidence: 99%

A simple isotopic labeling method to study cysteine oxidation in Alzheimer’s disease: oxidized cysteine-selective dimethylation (OxcysDML)

Robinson

2016

Anal Bioanal Chem

View full text Add to dashboard Cite

Cysteine is widely involved in redox signaling pathways through a number of reversible and irreversible modifications. Reversible modifications (e.g., S-glutathionylation, S-nitrosylation, disulfide bonds, and sulfenic acid) are used to protect proteins from oxidative attack and maintain cellular homeostasis, while irreversible oxidations (e.g., sulfinic acid and sulfonic acid) serve as hallmarks of oxidative stress. Proteomic analysis of cysteine-enriched peptides coupled with reduction of oxidized thiols can be used to measure the oxidation states of cysteine, which is helpful for elucidating the role that oxidative stress plays in biology and disease. As an extension of our previously reported cysDML method, we have developed oxidized cysteine-selective dimethylation (OxcysDML), to investigate the site-specific total oxidation of cysteine residues in biologically relevant samples. OxcysDML employs (1) blocking of free thiols by a cysteine-reactive reagent, (2) enrichment of peptides containing reversibly oxidized cysteine by a solid phase resin, and (3) isotopic labeling of peptide amino groups to quantify cysteine modifications arising from different biological conditions. On-resin enrichment and labeling minimizes sample handing time and improves efficiency in comparison with other redox proteomic methods. OxcysDML is also inexpensive and flexible, as it can accommodate the exploration of various cysteine modifications. Here, we applied the method to liver tissues from a late-stage Alzheimer's disease (AD) mouse model and wild-type (WT) controls. Because we have previously characterized this proteome using the cysDML approach, we are able here to probe deeper into the redox status of cysteine in AD. OxcysDML identified 1129 cysteine sites (from 527 proteins), among which 828 cysteine sites underwent oxidative modifications. Nineteen oxidized cysteine sites had significant alteration levels in AD and represent proteins involved in metabolic processes. Overall, we have demonstrated OxcysDML as a simple, rapid, robust, and inexpensive redox proteomic approach that is useful for gaining deeper insight into the proteome of AD.

show abstract

Section: Resultsmentioning

confidence: 99%

A simple isotopic labeling method to study cysteine oxidation in Alzheimer’s disease: oxidized cysteine-selective dimethylation (OxcysDML)

Robinson

2016

Anal Bioanal Chem

View full text Add to dashboard Cite

show abstract

“…The supernatant solutions were saved, and proteins were precipitated by adding 4× excess volumes of ice-cold precipitation solvents (acetone:ethanol: acetic acid = 50:50:0.1). After centrifugation, the protein pellet was redissolved in a solution with 8 M urea and 50 mM Hepes (pH 8) (91).…”

Section: Methodsmentioning

confidence: 99%

Targeting cancer cell integrins using gold nanorods in photothermal therapy inhibits migration through affecting cytoskeletal proteins

Ali

Tang

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

162

113

View full text Add to dashboard Cite

Metastasis is responsible for most cancer-related deaths, but the current clinical treatments are not effective. Recently, gold nanoparticles (AuNPs) were discovered to inhibit cancer cell migration and prevent metastasis. Rationally designed AuNPs could greatly benefit their antimigration property, but the molecular mechanisms need to be explored. Cytoskeletons are cell structural proteins that closely relate to migration, and surface receptor integrins play critical roles in controlling the organization of cytoskeletons. Herein, we developed a strategy to inhibit cancer cell migration by targeting integrins, using Arg-Gly-Asp (RGD) peptide-functionalized gold nanorods. To enhance the effect, AuNRs were further activated with 808-nm near-infrared (NIR) light to generate heat for photothermal therapy (PPTT), where the temperature was adjusted not to affect the cell viability/proliferation. Our results demonstrate changes in cell morphology, observed as cytoskeleton protrusions-i.e., lamellipodia and filopodia-were reduced after treatment. The Western blot analysis indicates the downstream effectors of integrin were attracted toward the antimigration direction. Proteomics results indicated broad perturbations in four signaling pathways, Rho GTPases, actin, microtubule, and kinases-related pathways, which are the downstream regulators of integrins. Due to the dominant role of integrins in controlling cytoskeleton, focal adhesion, actomyosin contraction, and actin and microtubule assembly have been disrupted by targeting integrins. PPTT further enhanced the remodeling of cytoskeletal proteins and decreased migration. In summary, the ability of targeting AuNRs to cancer cell integrins and the introduction of PPTT stimulated broad regulation on the cytoskeleton, which provides the evidence for a potential medical application for controlling cancer metastasis.gold nanorods | cytoskeleton | integrin | metastasis | plasmonic photothermal therapy

show abstract

“…Compared to label-free quantitative techniques, isotopelabeling methods can achieve the robust, accurate and high throughput proteome quantification by introducing different mass tags to peptides or proteins [68]. In terms of the incorporation of isotopic mass tags, isotope-labeling methods can have the enzymatic labeling, metabolic labeling, and chemical labeling.…”

Section: Quantitative Proteomic With Labeling Methodsmentioning

confidence: 99%

“…Wu et al [68] extended the conventional dual or triple dimethyl labeling to five-plex at the full MS scan (MS1) level without changing the handling procedures for the first time. In their research, Lys-C was used to digest protein samples, so the generated peptides contain at least two labeling sites (N-terminus and ε-amino group of lysine).…”

Section: Chemical Labelingmentioning

confidence: 99%

Proteomic profiling of protein corona formed on the surface of nanomaterial

Zhang

2015

Sci. China Chem.

View full text Add to dashboard Cite

The formation of protein coronas on nanomaterial will significantly alter the surface properties of nanomaterial in biological systems and subsequently impact biological responses including signaling, cellular uptake, transport, and toxicity etc. It is of critical importance to understand the formation of protein coronas on the surface of nanomaterial. Analytical techniques, especially mass spectrometry-based proteomics methods, are playing a key role for the qualitative and quantitative analyses of protein coronas on nanomaterial. In this review, the proteomic approaches developed for the characterization of protein coronas on various nanomaterials are introduced with the emphasis on the mass spectrometry-based proteomic strategies.protein corona, nanomaterial, proteomics, mass spectrometry, identification and quantification, gel electrophoresis

show abstract

Five-plex isotope dimethyl labeling for quantitative proteomics

Abstract: Stable isotope dimethyl labeling, a widely used method for quantitative proteomics, was extended to five channels for the first time. Comprehensive proteome and phosphoproteome quantification validated the high quantification accuracy and throughput of this five-plex method.

Cited by 52 publications

References 16 publications

A simple isotopic labeling method to study cysteine oxidation in Alzheimer’s disease: oxidized cysteine-selective dimethylation (OxcysDML)

A simple isotopic labeling method to study cysteine oxidation in Alzheimer’s disease: oxidized cysteine-selective dimethylation (OxcysDML)

Targeting cancer cell integrins using gold nanorods in photothermal therapy inhibits migration through affecting cytoskeletal proteins

Proteomic profiling of protein corona formed on the surface of nanomaterial

Contact Info

Product

Resources

About