Multidimensional separation of tryptic peptides from human serum proteins using reversed-phase, strong cation exchange, weak anion exchange, and fused-core fluorinated stationary phases

Boichenko, Alexander P.; Govorukhina, Natalia; Zee, Ate G.J. van der; Bischoff, Rainer

doi:10.1002/jssc.201300750

Cited by 26 publications

(23 citation statements)

References 41 publications

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“…ERLIC has been reported to separate phosphopeptides relatively evenly across fractions, permitting higher numbers of phosphopeptide identifications (34,35). Consistent with these studies, we observed that phosphopeptides separated quite evenly across the 24 ERLIC fractions (Fig.…”

Section: V600esupporting

confidence: 89%

A Phosphoproteomic Comparison of B-RAFV600E and MKK1/2 Inhibitors in Melanoma Cells*

Stuart¹,

Houel²,

Lee³

et al. 2015

Molecular & Cellular Proteomics

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Inhibitors of oncogenic B-RAFV600E and MKK1/2 have yielded remarkable responses in B-RAF V600E -positive melanoma patients. However, the efficacy of these inhibitors is limited by the inevitable onset of resistance. Despite the fact that these inhibitors target the same pathway, combination treatment with B-RAF V600E and MKK1/2 inhibitors has been shown to improve both response rates and progression-free survival in B-RAF V600E melanoma patients. To provide insight into the molecular nature of the combinatorial response, we used quantitative mass spectrometry to characterize the inhibitor-dependent phosphoproteome of human melanoma cells treated with the B-RAF V600E inhibitor PLX4032 (vemurafenib) or the MKK1/2 inhibitor AZD6244 (selumetinib). In three replicate experiments, we quantified changes at a total of 23,986 phosphosites on 4784 proteins. This included 1317 phosphosites that reproducibly decreased in response to at least one inhibitor. Phosphosites that responded to both inhibitors grouped into networks that included the nuclear pore complex, growth factor signaling, and transcriptional regulators. Although the majority of phosphosites were responsive to both inhibitors, we identified 16 sites that decreased only in response to PLX4032, suggesting rare instances where oncogenic B-RAF signaling occurs in an MKK1/2-independent manner. Only two phosphosites were identified that appeared to be uniquely responsive to AZD6244. When cells were treated with the combination of AZD6244 and PLX4032 at subsaturating concentrations (30 nM), responses at nearly all phosphosites were additive. We conclude that AZD6244 does not substantially widen the range of phosphosites inhibited by PLX4032 and that the benefit of the drug combination is best explained by their additive effects on suppressing ERK1/2 signaling. Comparison of our results to another recent ERK1/2 phosphoproteomics study revealed a surprising degree of variability in the sensitivity of phosphosites to MKK1/2 inhibitors in human cell lines, revealing unexpected cell specificity in the molecular responses to pathway activation. Molecular & Cellular

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Section: V600esupporting

confidence: 89%

A Phosphoproteomic Comparison of B-RAFV600E and MKK1/2 Inhibitors in Melanoma Cells*

Stuart¹,

Houel²,

Lee³

et al. 2015

Molecular & Cellular Proteomics

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“…Boichenko et al [43] reported multidimensional separation of tryptic peptides from human serum proteins. The study demonstrated that peptide fractionation on SCX, weak anion exchange (WAX) in the electrostatic repulsion hydrophilic interaction chromatography (ERLIC) mode, RP C18 at pH 2.5 (low pH), fused-core fluorinated at pH 2.5, and RP C18 at pH 9.7 (high pH) stationary phases resulted in two to three times more identified proteins and three to four times more identified peptides in comparison with 1D nano Chip-LC–MS/MS quadrupole TOF analysis.…”

Section: Methodologies Used To Reduce the Complexity Of Proteomic Smentioning

confidence: 99%

Liquid phase based separation systems for depletion, prefractionation, and enrichment of proteins in biological fluids and matrices for in‐depth proteomics analysis—An update covering the period 2011–2014

2014

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This review article expands on the previous one (S. Selvaraju and Z. El Rassi, Electrophoresis 2012, 33, 74-88) by reviewing pertinent literature in the period extending from early 2011 to present. As the previous review article, the present one is concerned with proteomic sample preparation (e.g., depletion of high abundance proteins, reduction of the protein dynamic concentration range, enrichment of a particular sub-proteome), and the subsequent chromatographic and/or electrophoretic pre-fractionation prior to peptide separation and identification by LC-MS/MS. This review article is distinguished from its second version published in Electrophoresis 2012, 33, 74-88 by expanding on capturing/enriching sub-phosphoproteomes by immobilized metal affinity chromatography and metal oxide affinity chromatography. Seventy-seven papers published in the period extending from mid 2011 to the present have been reviewed. By no means this review article is exhaustive, given the fact that its aim is to give a concise treatment of the latest developments in the field.

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“…20 The second dimension involved a nanoChip-LC (RP C18, pH 2.6) and Q TOF-MS/MS. The highest number of peptides was identified by ERLIC, while an equal number of proteins were identified by ERLIC and high-pH RPLC.…”

Section: De Jong Et Al Compared Erlic-ms Directly With Rplc-ms For Smentioning

confidence: 99%

“…With this combination there is a uniform distribution of peptides among the collected fractions, with peptides eluting in order of decreasing isoelectric point and increasing hydrophilicity. [18][19][20] This order of elution is comparable in some respects to that of isoelectric focusing but is accomplished without ampholines or a complex mixture of buffering salts. The separation of peptides based on isoelectric point has been exploited for the study of protein/ peptide deamidation 21,22 as the original residue, Asn, and its deamidated products n-Asp and isoAsp have different pKa values.…”

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confidence: 99%

The Use of Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC) for Proteomics Research

Ng¹,

Hao²,

Sze³

2014

Mass Spectrometry Letters

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Characterization and studies of proteome are challenging because biological samples are complex, with a wide dynamic range of abundance. At present the proteins are identified by digestion into peptides, with subsequent identification of the peptides by mass spectrometry (MS). MS is a powerful technique for the purpose, but it cannot identify every peptide in such complex mixtures simultaneously. For accurate analysis and quantification it is important to separate the peptides first by chromatography into fractions of a size that MS can handle. With these less complex fractions, the probability is increased of identifying peptides of low abundance that would otherwise experience ion suppression effects due to the presence of peptides of high abundance. Enrichment for peptides with certain post-translational modifications helps to increase their detection rates as well. Electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) is a mixed-mode chromatographic technique which combines the use of electrostatic repulsion and hydrophilic interaction. This review provides an overview of ERLIC and its various proteomics applications. ERLIC has been demonstrated to have good orthogonality to reverse phase liquid chromatography (RPLC), making it useful as a first dimension in multidimensional liquid chromatography (MDLC) and fractionation of digests in general. Peptides elute in order of their isoelectric points and polarity. ERLIC has also been successfully utilized for the enrichment for phosphopeptides and glycopeptides, facilitating their identification. In addition, it is promising for the study of peptide deamidation. ERLIC performs comparably well or better than established methods for these various applications, and serves as a viable and efficient workflow alternative.

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Multidimensional separation of tryptic peptides from human serum proteins using reversed-phase, strong cation exchange, weak anion exchange, and fused-core fluorinated stationary phases

Cited by 26 publications

References 41 publications

A Phosphoproteomic Comparison of B-RAFV600E and MKK1/2 Inhibitors in Melanoma Cells*

A Phosphoproteomic Comparison of B-RAFV600E and MKK1/2 Inhibitors in Melanoma Cells*

Liquid phase based separation systems for depletion, prefractionation, and enrichment of proteins in biological fluids and matrices for in‐depth proteomics analysis—An update covering the period 2011–2014

The Use of Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC) for Proteomics Research

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