Automated metal-free multiple-column nanoLC for improved phosphopeptide analysis sensitivity and throughput

Zhao, Rui; Ding, Shi‐Jian; Shen, Yufeng; Camp, David G.; Livesay, Eric A.; Udseth, Harold R.; Smith, Richard D.

doi:10.1016/j.jchromb.2008.12.068

Cited by 29 publications

(30 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Samples were analyzed using a LTQ Orbitrap Velos MS (Thermo Scientific) coupled to an automated dual-column metal-free nanoLC platform (49). Details of the separation and mass spectrometer parameters are described in SI Methods.…”

Section: Methodsmentioning

confidence: 99%

Tandem mass spectrometry identifies many mouse brain O -GlcNAcylated proteins including EGF domain-specific O -GlcNAc transferase targets

Alfaro

Gong

Monroe

et al. 2012

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

Self Cite

264

265

View full text Add to dashboard Cite

O -linked N -acetylglucosamine ( O -GlcNAc) is a reversible posttranslational modification of Ser and Thr residues on cytosolic and nuclear proteins of higher eukaryotes catalyzed by O -GlcNAc transferase (OGT). O -GlcNAc has recently been found on Notch1 extracellular domain catalyzed by EGF domain-specific OGT. Aberrant O -GlcNAc modification of brain proteins has been linked to Alzheimer's disease (AD). However, understanding specific functions of O -GlcNAcylation in AD has been impeded by the difficulty in characterization of O -GlcNAc sites on proteins. In this study, we modified a chemical/enzymatic photochemical cleavage approach for enriching O -GlcNAcylated peptides in samples containing ∼100 μg of tryptic peptides from mouse cerebrocortical brain tissue. A total of 274 O -GlcNAcylated proteins were identified. Of these, 168 were not previously known to be modified by O -GlcNAc. Overall, 458 O -GlcNAc sites in 195 proteins were identified. Many of the modified residues are either known phosphorylation sites or located proximal to known phosphorylation sites. These findings support the proposed regulatory cross-talk between O -GlcNAcylation and phosphorylation. This study produced the most comprehensive O -GlcNAc proteome of mammalian brain tissue with both protein identification and O -GlcNAc site assignment. Interestingly, we observed O -β-GlcNAc on EGF-like repeats in the extracellular domains of five membrane proteins, expanding the evidence for extracellular O -GlcNAcylation by the EGF domain-specific OGT. We also report a GlcNAc-β-1,3-Fuc-α-1- O -Thr modification on the EGF-like repeat of the versican core protein, a proposed substrate of Fringe β-1,3- N -acetylglucosaminyltransferases.

show abstract

Section: Methodsmentioning

confidence: 99%

Tandem mass spectrometry identifies many mouse brain O -GlcNAcylated proteins including EGF domain-specific O -GlcNAc transferase targets

Alfaro

Gong

Monroe

et al. 2012

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

Self Cite

264

265

View full text Add to dashboard Cite

show abstract

“…Each sample was manually loaded using a pressure cell (Brechbuhler Inc., Houston, TX), the nano-LC-ESI column/tip was connected to the LC system, and the postsplit flow rate was manually calibrated to be 350 nL/min (ϳ70 bar, ϳ70 l/min presplit) using a calibrated micropipette. Note that because the sample was manually loaded in this manner, the phosphopeptides were never exposed to metal or other surfaces that might have caused sample loss (39). A 2 kV ESI voltage was applied and the ESI spray was observed.…”

Section: Methodsmentioning

confidence: 99%

Discovery of Mouse Spleen Signaling Responses to Anthrax using Label-Free Quantitative Phosphoproteomics via Mass Spectrometry

Manes

Dong

Zhou

et al. 2011

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

Inhalational anthrax is caused by spores of the bacteriumBacillus anthracis (B. anthracis), and is an extremely dangerous disease that can kill unvaccinated victims within 2 weeks. Modern antibiotic-based therapy can increase the survival rate to ϳ50%, but only if administered presymptomatically (within 24 -48 h of exposure). To discover host signaling responses to presymptomatic anthrax, labelfree quantitative phosphoproteomics via liquid chromatography coupled to mass spectrometry was used to compare spleens from uninfected and spore-challenged mice over a 72 h time-course. Spleen proteins were denatured using urea, reduced using dithiothreitol, alkylated using iodoacetamide, and digested into peptides using trypsin, and the resulting phosphopeptides were enriched using titanium dioxide solid-phase extraction and analyzed by nano-liquid chromatography-Linear Trap Quadrupole-Orbitrap-MS(/MS). The fragment ion spectra were processed using DeconMSn and searched using both Mascot and SEQUEST resulting in 252,626 confident identifications of 6248 phosphopeptides (corresponding to 5782 phosphorylation sites). The precursor ion spectra were deisotoped using Decon2LS and aligned using MultiAlign resulting in the confident quantitation of 3265 of the identified phosphopeptides. ANOVAs were used to produce a q-value ranked list of host signaling responses. Late-stage (48 -72 h postchallenge) Sterne strain (lethal) infections resulted in global alterations to the spleen phosphoproteome. In contrast, ⌬Sterne strain (asymptomatic; missing the anthrax toxin) infections resulted in 188 (5.8%) significantly altered (q<0.05) phosphopeptides. Twenty-six highly tentative phosphorylation responses to early-stage (24 h postchallenge) anthrax were discovered (q<0.5), and ten of these originated from eight proteins that have known roles in the host immune response. These tentative early-anthrax host response signaling events within mouse spleens may translate into presymptomatic diagnostic biomarkers of human anthrax detectable within circulating immune cells, and could aid in the identification of pathogenic mechanisms and therapeutic targets.

show abstract

“…The automated metal-free nano LC system, which was specifically developed to obtain broad and sensitive analysis of phosphopeptides, is described in detail in (22). MS data were acquired for 200 min, beginning 85 min after sample injection (30 min into gradient).…”

Section: Materials-trizolmentioning

confidence: 99%

“…In the current study, we report differential protein phosphorylation in soybean root hair cells in response to B. japonicum interaction enabled by the use of ultra-sensitive phosphopeptide LC platform coupled with high-performance MS (22). We employed eight-plex iTRAQ for relative quantification of phosphopeptides in root hairs and stripped roots across nine time points during the first 48 h after rhizobial inoculation.…”

mentioning

confidence: 99%

Quantitative Phosphoproteomic Analysis of Soybean Root Hairs Inoculated with Bradyrhizobium japonicum

Nguyen

Brechenmacher

Aldrich

et al. 2012

Molecular & Cellular Proteomics

123

View full text Add to dashboard Cite

Root hairs are single hair-forming cells on roots that function to increase root surface area, enhancing water and nutrient uptake. In leguminous plants, root hairs also play a critical role as the site of infection by symbiotic nitrogen fixing rhizobia, leading to the formation of a novel organ, the nodule. The initial steps in the rhizobia-root hair infection process are known to involve specific receptor kinases and subsequent kinase cascades. Here, we characterize the phosphoproteome of the root hairs and the corresponding stripped roots (i.e. roots from which root hairs were removed) during rhizobial colonization and infection to gain insight into the molecular mechanism of root hair cell biology. We chose soybean (Glycine max L.), one of the most important crop plants in the legume family, for this study because of its larger root size, which permits isolation of sufficient root hair material for phosphoproteomic analysis. Phosphopeptides derived from root hairs and stripped roots, mock inoculated or inoculated with the soybean-specific rhizobium Bradyrhizobium japonicum, were labeled with the isobaric tag eightplex iTRAQ, enriched using Ni-NTA magnetic beads and subjected to nanoRPLC-MS/MS 1 analysis using HCD and decision tree guided CID/ETD strategy. Root hairs are known to play an important role in increasing the root surface area for water and nutrient uptake from the soil (1). Found on the surface of the maturation zone of primary and secondary roots, root hairs develop from specialized epidermal cells (trichoblast). New root hair cells continuously develop in the elongation zone, elongating and maturing as the root grows (2). In addition to the critical role in nutrient uptake, the root hair is the primary infection site for symbiotic bacteria (rhizobia) in legume plants. During the first stages of the legume-rhizobium interaction, (iso)flavonoids secreted by the legume induce the rhizobia to synthesize the Nod factor, a specific lipo-chito-oligosaccharide. This bacterial signal molecule elicits a variety of very rapid (within minutes) responses in the root hair cell, including depolarization of the membrane potential and induction of calcium oscillations (3). The root hair then curls to form a shepherd's crook structure, where the rhizobia become entrapped within the root hair cell wall (4), leading subsequently to an invagination of the root hair plasma membrane and the formation of the tubular infection thread structure by which the bacteria ultimately gain access to the root cortex (5).Root hair physiology under both controlled and biotic/abiotic stress conditions has been studied intensively using a variety of approaches. Numerous root hair mutants have been identified and subsequently linked to the function of various 1 The abbreviations used are: nanoRPLC-MS/MS, nano reversedphase liquid chromatography-tandem mass spectrometry; HCD-CID/ ETD, high energy/collision-induced dissociation/electron transfer dissociation; Ni-NTA, Nickel-nitriloacetic acid; iTRAQ, Isobaric tag for relative and absolu...

show abstract

Automated metal-free multiple-column nanoLC for improved phosphopeptide analysis sensitivity and throughput

Cited by 29 publications

References 31 publications

Tandem mass spectrometry identifies many mouse brain O -GlcNAcylated proteins including EGF domain-specific O -GlcNAc transferase targets

Tandem mass spectrometry identifies many mouse brain O -GlcNAcylated proteins including EGF domain-specific O -GlcNAc transferase targets

Discovery of Mouse Spleen Signaling Responses to Anthrax using Label-Free Quantitative Phosphoproteomics via Mass Spectrometry

Quantitative Phosphoproteomic Analysis of Soybean Root Hairs Inoculated with Bradyrhizobium japonicum

Contact Info

Product

Resources

About