A fully integrated multi-column system for abundant protein depletion from serum/plasma

Janecki, Dariusz; Pomerantz, Steven C.; Beil, E.; Nemeth, Jennifer F.

doi:10.1016/j.jchromb.2012.06.010

Cited by 10 publications

(8 citation statements)

References 29 publications

(37 reference statements)

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“…To avoid RBC lysis, blood samples were immediately centrifuged at 2000 x g for 10 minutes. The plasma was collected into 100uL aliquots and stored at -80 o C. Plasma samples were depleted of high-and mid-abundant proteins using immune-depletion columns (Agilent Multiple Affinity Removal Column MARS3 (Agilent 5188-5218) coupled to the Seppro mouse LC5 SuperMix column (Sigma S5824) or Seppro mouse LC10 IgY7 column (Sigma S5699) coupled to a Seppro mouse LC5 SuperMix column) on an ÄKTA HPLC system 48 . Protein concentrations before and after depletion were measured by BCA Protein Assay Kit (ThermoFisher, 23235).…”

Section: Discussionmentioning

confidence: 99%

“…Each sample pool was further divided into four sub-pools and these sub-pools were randomized across the depletion process to reduce the chance of introducing batch effects. Plasma samples were depleted once each using human IgY14 LC10 (Sigma S5074) and human Supermix LC5 (Sigma S5324) columns coupled to an ÄKTA HPLC system 48 . Independent depleted plasma samples were collected in a single 20 mL flowthrough fraction, pooled by sub-pool, concentrated with an Amicon Ultra Centrifugal Filter Units (3 kDa cutoff, Millipore UFC900324) and buffer exchanged with 50 mM Ammonium bicarbonate (Sigma A6141).…”

Section: Data Analysis For Identification Of Candidate Biomarkers Raw Ms/ms Spectra From the Analysismentioning

confidence: 99%

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Multiplexed triage of candidate biomarkers in plasma using internal standard triggered-parallel reaction monitoring mass spectrometry

Kennedy

Whiteaker

Ivey

et al. 2021

Preprint

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Despite advances in proteomic technologies, clinical translation of plasma biomarkers remains low, partly due to a major bottleneck between the discovery of candidate biomarkers and downstream costly clinical validation studies. Due to a dearth of multiplexable assays, generally only a few candidate biomarkers are tested, and the validation success rate is accordingly low. Here, we demonstrate the capability of internal standard triggered-parallel reaction monitoring (IS-PRM) to prioritize candidate biomarkers for validation studies. A 5,176-plex assay coupling immunodepletion and fractionation with IS-PRM was developed and implemented in human plasma to quantify peptides representing 1,314 breast cancer biomarker candidates. Compared to prior approaches using data-dependent analysis, IS-PRM showed improved sensitivity (912 vs 295 proteins quantified) and precision (CV 0.1 vs 0.27) enabling rank-ordering of candidate biomarkers for validation studies. The assay greatly expands capabilities for quantification of large numbers of proteins and is well suited for prioritization of viable candidate biomarkers.

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Section: Discussionmentioning

confidence: 99%

Section: Data Analysis For Identification Of Candidate Biomarkers Raw Ms/ms Spectra From the Analysismentioning

confidence: 99%

Multiplexed triage of candidate biomarkers in plasma using internal standard triggered-parallel reaction monitoring mass spectrometry

Kennedy

Whiteaker

Ivey

et al. 2021

Preprint

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“…Janecki et al [6] developed a multi-column system for the depletion of abundant proteins. This involved the transformation of a conventional HPLC system to a low backpressure liquid chromatography set-up for automated serum/plasma depletion and fractionation.…”

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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“…Because biologically relevant glycoproteins are frequently present at relatively low levels in complex biological samples, and can be masked by higher abundance proteins in the sample, prefractionation is often mandatory. One of the most commonly employed strategies consists of removal of high abundance proteins, including albumin and immunoglobulins that interfere with the detection of less abundant proteins . Alternatively, samples can be fractionated by chromatographic or other means of separation, to further reduce complexity and enrich glycoproteins prior to MS .…”

Section: Glycoprotein Enrichment Strategies For Proteomics Analysismentioning

confidence: 99%

Glycocapture‐based proteomics for secretome analysis

2013

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Protein glycosylation represents the most abundant extracellular posttranslational modification in multicellular organisms. These glycoproteins unequivocally comprise the major biomolecules involved in extracellular processes, such as growth factors, signaling proteins for cellular communication, enzymes, and proteases for on- and off-site processing. It is now known that altered protein glycosylation is a hallmark event in many different pathologies. Glycoproteins are found mostly in the so-called secretome, which comprises classically and nonclassically secreted proteins and protein fragments that are released from the cell surface through ectodomain shedding. Due to biological complexity and technical difficulty, comparably few studies have taken an in-depth investigation of cellular secretomes using system-wide approaches. The cellular secretomes are considered to be a valuable source of therapeutic targets and novel biomarkers. It is not surprising that many existing biomarkers, including biomarkers for breast, ovarian, prostate, and colorectal cancers are glycoproteins. Focused analysis of secreted glycoproteins could thus provide valuable information for early disease diagnosis, and surveillance. Furthermore, since most secreted proteins are glycosylated and glycosylation predominantly targets secreted proteins, the glycan/sugar moiety itself can be used as a chemical "handle" for the targeted analysis of cellular secretomes, thereby reducing sample complexity and allowing detection of low abundance proteins in proteomic workflows. This review will focus on various glycoprotein enrichment strategies that facilitate proteomics-based technologies for the quantitative analysis of cell secretomes and cell surface proteomes.

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A fully integrated multi-column system for abundant protein depletion from serum/plasma

Cited by 10 publications

References 29 publications

Multiplexed triage of candidate biomarkers in plasma using internal standard triggered-parallel reaction monitoring mass spectrometry

Multiplexed triage of candidate biomarkers in plasma using internal standard triggered-parallel reaction monitoring mass spectrometry

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

Glycocapture‐based proteomics for secretome analysis

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