Platform for Establishing Interlaboratory Reproducibility of Selected Reaction Monitoring-Based Mass Spectrometry Peptide Assays

Prakash, Amol; Rezai, Taha; Krastins, Bryan; Sarracino, David; Athanas, Michael; Russo, Paul; Ross, Mark M.; Zhang, H.; Tian, Yuan; Kulasingam, Vathany; Drabovich, Andrei P.; Smith, Christopher R.; Batruch, Ihor; Liotta, Lance A.; Petricoin, Emanuel F.; Diamandis, Eleftherios P.; Chan, Daniel W.; Lopez, Mary F.

doi:10.1021/pr100821m

Cited by 72 publications

(70 citation statements)

References 35 publications

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“…Despite possible new sources of imprecision introduced by these additional steps, still acceptable intra-and inter-laboratory CVs usually below 12% were obtained at mid-point of the linear range even after applying peptide fractionation or immuno-enrichment (28,29,31,32).…”

Section: Discussionmentioning

confidence: 99%

Total ApoE and ApoE4 Isoform Assays in an Alzheimer's Disease Case-control Study by Targeted Mass Spectrometry (n = 669): A Pilot Assay for Methionine-containing Proteotypic Peptides

Simon

Girod

Fonbonne

et al. 2012

Molecular & Cellular Proteomics

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Allelic polymorphism of the apolipoprotein E (ApoE) gene (ApoE 2, ApoE 3 and ApoE 4 alleles) gives rise to three protein isoforms (ApoE2, ApoE3 and ApoE4) that differ by 1 or 2 amino acids. Inheritance of the ApoE 4 allele is a risk factor for developing Alzheimer's disease (AD). The potential diagnostic value of ApoE protein levels in biological fluids (i.e. cerebrospinal fluid, plasma and serum) for distinguishing between AD patients and healthy elderly subjects is subject to great controversy. Although a recent study reported subnormal total ApoE and ApoE4 levels in the plasma of AD patients, other studies have found normal or even elevated protein levels (versus controls). Because all previously reported assays were based on immunoenzymatic techniques, we decided to develop an orthogonal assay based on targeted mass spectrometry by tracking (i) a proteotypic peptide common to all ApoE isoforms and (ii) a peptide that is specific for the 4 allele. After trypsin digestion, the ApoE4-specific peptide contains an oxidation-prone methionine residue. The endogenous methionine oxidation level was evaluated in a small cohort (n ‫؍‬ 68) of heterozygous 34 carriers containing both healthy controls and AD patients. As expected, the proportion of oxidized residues varied from 0 to 10%, with an average of 5%. We therefore developed a standardized strategy for the unbiased, absolute quantification of ApoE4, based on performic acid oxidization of methionine. Once the sample workflow had been thoroughly validated, it was applied to the concomitant quantification of total ApoE and ApoE4 isoform in a large casecontrol study (n ‫؍‬ 669). The final measurements were consistent with most previously reported ApoE concentration values and confirm the influence of the different alleles on the protein expression level. Our results illustrate (i) the reliability of selected reaction monitoringbased assays and (ii) the value of the oxidization step for unbiased monitoring of methionine-containing proteotypic peptides. Furthermore, a statistical analysis indicated that neither total ApoE and ApoE4 levels nor the ApoE/ApoE4 ratio correlated with the diagnosis of AD. These findings reinforce the conclusions of previous studies in which plasma ApoE levels had no obvious clinical significance. Molecular & Cellular

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Section: Discussionmentioning

confidence: 99%

Total ApoE and ApoE4 Isoform Assays in an Alzheimer's Disease Case-control Study by Targeted Mass Spectrometry (n = 669): A Pilot Assay for Methionine-containing Proteotypic Peptides

Simon

Girod

Fonbonne

et al. 2012

Molecular & Cellular Proteomics

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“…Additionally, very little is known about the adaptive changes that occur following the inhibition of MEK/ERK signaling in NRAS mutant melanoma. To address these issues, the optimal technique is liquid chromatography-multiple reaction monitoring mass spectrometry, which been shown to be highly reproducible and portable across laboratories (15)(16)(17)(18).…”

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confidence: 99%

Evaluating Melanoma Drug Response and Therapeutic Escape with Quantitative Proteomics

Rebecca

Wood

Fedorenko

et al. 2014

Molecular & Cellular Proteomics

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The evolution of cancer therapy into complex regimens with multiple drugs requires novel approaches for the development and evaluation of companion biomarkers. Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) is a versatile platform for biomarker measurement. In this study, we describe the development and use of the LC-MRM platform to study the adaptive signaling responses of melanoma cells to inhibitors of HSP90 (XL888) and MEK (AZD6244). XL888 had good anti-tumor activity against NRAS mutant melanoma cell lines as well as BRAF mutant cells with acquired resistance to BRAF inhibitors both in vitro and in vivo. LC-MRM analysis showed HSP90 inhibition to be associated with decreased expression of multiple receptor tyrosine kinases, modules in the PI3K/AKT/mammalian target of rapamycin pathway, and the MAPK/CDK4 signaling axis in NRAS mutant melanoma cell lines and the inhibition of PI3K/AKT signaling in BRAF mutant melanoma xenografts with acquired vemurafenib resistance. The LC-MRM approach targeting more than 80 cancer signaling proteins was highly sensitive and could be applied to fine needle aspirates from xenografts and clinical melanoma specimens (using 50 g of total protein). We further showed MEK inhibition to be associated with signaling through the NFB and WNT signaling pathways, as well as increased receptor tyrosine kinase expression and activation. Validation studies identified PDGF receptor ␤ signaling as a potential escape mechanism from MEK inhibition, which could be overcome through combined use of AZD6244 and the PDGF receptor inhibitor, creno- Despite excitement about the development of targeted therapy strategies for cancer, few cures have been achieved. In patients with BRAF mutant melanoma, treatment with small molecule BRAF inhibitors typically follows a course of response and tumor shrinkage followed by eventual relapse and resistance (mean progression-free survival is ϳ5.3 months) (1). Resistance to BRAF inhibitors is typically accompanied by reactivation of the MAPK signaling pathway, an effect mediated through activating mutations in NRAS and MEK1/2, genomic amplification of BRAF, increased expression of CRAF and Cot, and the acquisition of BRAF splice-form mutants (2-5). There is also evidence that increased PI3K/AKT signaling, resulting from the genetic inactivation of PTEN and NF1 and increased receptor tyrosine kinase (RTK) 1 signaling, may be involved in acquired BRAF inhibitor resistance (5-7). Many of the signaling proteins implicated in the escape from BRAF inhibitor therapy are clients of heat shock protein (HSP)-90 (8). Preclinical evidence now indicates that HSP90 inhibitors can overcome acquired and intrinsic BRAF inhibitor resistance, and clinical trials have been initiated to evaluate the BRAF/HSP90 combination in newly diagnosed patients (8, 9).Although targeted therapy strategies have been promising in BRAF mutant melanoma, few options currently exist for the 15-20% of melanoma patients whose tumors harbor activating NRAS mutations (10). Alth...

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“…The proteome of SP is as complex as the proteome of serum and contains large amounts of semenogelins, PSA and other high-abundance proteins 18,20,21 . We previously identified more than 3,000 proteins in SP of healthy men and patients with infertility, prostatitis and prostate cancer 21,22,56 .…”

Section: Discussionmentioning

confidence: 99%

Multi-omics biomarker pipeline reveals elevated levels of protein-glutamine gamma-glutamyltransferase 4 in seminal plasma of prostate cancer patients

Drabovich

Saraon

Drabovich³

et al. 2017

Preprint

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Prostate-specific antigen, a widely used biomarker of prostate cancer, lacks specificity and prognostic significance, which often results in over-diagnosis and over-treatment of prostate cancer. In this study, we investigated if seminal plasma proteins could increase specificity of detecting primary prostate cancer and discriminate between low-and high-grade cancers. To select 148 most promising biomarker candidates, we combined transcripts or proteins identified through five independent data mining or experimental approaches: tissue transcriptomics, seminal plasma proteomics, cell secretomics, tissue specificity and androgen regulation. To follow up these candidates, we designed a rigorous biomarker verification and validation pipeline based on multiplex quantitative selected reaction monitoring assays. We verified 77 and validated 19 most promising proteins in seminal plasma of 67 negative biopsy and 152 prostate cancer patients. Validation revealed a prostate-specific, secreted and androgen-regulated protein-glutamine gamma-glutamyltransferase 4 (TGM4), which could predict prostate cancer on biopsy and outperformed age and serum PSA. In the independent validation set re-measured by an in-house ELISA, TGM4 was significantly up-regulated (3.7-fold, P=0.005) and revealed AUC 0.66 for differentiating between negative biopsy and prostate cancer in patients with age ≥50 and blood serum PSA ≥4 ng/mL. Interestingly, none of validated biomarkers could differentiate between low-and high-grade cancers. Very low levels of TGM4 (120 pg/mL) were detected by ELISA in blood serum, but could not differentiate between negative biopsy, prostate cancer or prostate inflammation. Significantly higher levels of TGM4 in blood serum (3.5-fold, P = 0.0004) were found for younger men (<40 y.o.) relative to older men (≥70 y.o.). Our work may represent one of the most comprehensive studies on prostate cancer biomarkers in seminal plasma. Even though moderate performance of TGM4 as a single biomarker will unlikely result in its immediate use in the clinic, TGM4 may be further investigated in the distinct subtypes of prostate cancer and included into emerging multibiomarker panels.

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Platform for Establishing Interlaboratory Reproducibility of Selected Reaction Monitoring-Based Mass Spectrometry Peptide Assays

Cited by 72 publications

References 35 publications

Total ApoE and ApoE4 Isoform Assays in an Alzheimer's Disease Case-control Study by Targeted Mass Spectrometry (n = 669): A Pilot Assay for Methionine-containing Proteotypic Peptides

Total ApoE and ApoE4 Isoform Assays in an Alzheimer's Disease Case-control Study by Targeted Mass Spectrometry (n = 669): A Pilot Assay for Methionine-containing Proteotypic Peptides

Evaluating Melanoma Drug Response and Therapeutic Escape with Quantitative Proteomics

Multi-omics biomarker pipeline reveals elevated levels of protein-glutamine gamma-glutamyltransferase 4 in seminal plasma of prostate cancer patients

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