Application of thermally assisted electrospray ionization mass spectrometry for detection of noncovalent complexes of bovine serum albumin with growth hormone releasing factor and other biologically active peptides

Baczynskyj, L.; Bronson, George E.; Kubiak, Teresa M.

doi:10.1002/rcm.1290080311

Cited by 21 publications

(10 citation statements)

References 21 publications

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“…This approach, which has the advantage of removing the most abundant proteins, also has disadvantages. For example, depletion targets can be carriers for other proteins and small molecules [30–34], which may variably travel with them in removal strategies, affecting quantification [26,27]. Conformational changes in antibody targets presumably affect epitope binding, resulting in altered depletion efficiencies.…”

Section: Discussionmentioning

confidence: 99%

Evaluating the effects of preanalytical variables on the stability of the human plasma proteome

Hassis

Niles

Braten

et al. 2015

Analytical Biochemistry

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High quality clinical biospecimens are vital for biomarker discovery, verification, and validation. Variations in blood processing and handling can affect protein abundances and assay reliability. Using an untargeted LC-MS approach, we systematically measured the impact of preanalytical variables on the plasma proteome. Time prior to processing was the only variable that affected the plasma protein levels. LC-MS quantification showed that preprocessing times <6 h had minimal effects on the immunodepleted plasma proteome, but by 4 days significant changes were apparent. Elevated levels of many proteins were observed, suggesting that in addition to proteolytic degradation during the preanalytical phase, changes in protein structure are also important considerations for protocols using antibody depletion. As to processing variables, a comparison of single- vs double-spun plasma showed minimal differences. After processing, the impact ≤3 freeze–thaw cycles was negligible regardless of whether freshly collected samples were processed in short succession or the cycles occurred during 14–17 years of frozen storage (−80 °C). Thus, clinical workflows that necessitate modest delays in blood processing times or employ different centrifugation steps can yield valuable samples for biomarker discovery and verification studies.

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Section: Discussionmentioning

confidence: 99%

Evaluating the effects of preanalytical variables on the stability of the human plasma proteome

Hassis

Niles

Braten

et al. 2015

Analytical Biochemistry

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“…Although albumin binds peptides such as insulin and bradykinin (31 ), the extent of this peptide Clinical Chemistry 53, No. 11, 2007 binding is unknown (32 ).…”

Section: Discussionmentioning

confidence: 99%

Quality Control of Serum Albumin Depletion for Proteomic Analysis

Seam¹,

Gonzales²,

Kern³

et al. 2007

Clinical Chemistry

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Background: Prefractionation techniques such as serum albumin depletion are useful precursors to proteomic analysis, but they may introduce preanalytical bias if the depletion is not reproducible. We examined the reproducibility of albumin immunodepletion and describe a method of QC for this process. Methods: Depletion of albumin from pooled serum, performed using IgY immunoaffinity spin columns, was assessed for 21 runs on each of 4 columns. We measured albumin concentrations, after albumin depletion, by use of an immunoturbidimetric assay on the Beckman LX 20 analyzer and assessed mass spectra of albumin-depleted samples by use of SELDI-TOF mass spectrometry. Results: There was substantial run-to-run variation in efficiency of albumin depletion, with systematic decline in efficiency after multiple uses of the columns. Mean depletion efficiency was >95% for 15 of the 1st 17 runs and <90% for runs 18 to 21. We evaluated the 10 highest-intensity peaks present in all spectra from runs 1, 8, 17, and 21 and assessed the effect of albumin depletion on SELDI-TOF mass spectrometry reproducibility. Comparing the %CV of relative intensities for low and high m/z measurements revealed a significant difference of run 21 compared with runs 1, 8, and 17 (P <0.0001). Six-fold more peaks were found in albumindepleted than unfractionated serum at both high and low m/z.

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“…Albumin is known to bind proteins, lipids, and small molecules including various peptide hormones (e.g., bradykinin, interferons, and insulin) and proteins (e.g., serum amyloid A and Streptococcal Protein G) [1][2][3][4][5][6]. Although albumin-binding peptides (below 30 kDa) in serum have been studied, the extent of their binding is currently unknown [7,8].…”

Section: Introductionmentioning

confidence: 99%

Investigation of an albumin‐enriched fraction of human serum and its albuminome

Gundry

Jelinek

et al. 2007

Proteomics Clinical Apps

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128

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The removal of albumin and other high abundance proteins is a routine first step in the analysis of serum and plasma proteomes. However, as albumin can bind proteins and peptides, there is a universal concern as to how the serum proteome is changed by the removal of albumin. To address this concern, the current study was designed to identify proteins and peptides removed from the serum during albumin depletion; to determine which of these are bound to albumin (rather than copurified) and whether the bound proteins are intact proteins or peptide fragments. Sequential, independent analyses including both anti-albumin antibody (anti-HSA) affinity chromatography and SEC were used to isolate albumin-bound proteins. RP-HPLC and 1-D SDS-PAGE were then used to further separate the proteins prior to identification by MS/MS. Finally, whole protein molecular weight (MW) MS measurements coupled with protein coverage obtained by MS were combined to assess whether the bound proteins were intact or peptide fragments. Combining the results from multiple approaches, 35 proteins, of which 24 are intact, were found to be associated with albumin, and they include both known high and low abundance proteins.

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Application of thermally assisted electrospray ionization mass spectrometry for detection of noncovalent complexes of bovine serum albumin with growth hormone releasing factor and other biologically active peptides

Cited by 21 publications

References 21 publications

Evaluating the effects of preanalytical variables on the stability of the human plasma proteome

Evaluating the effects of preanalytical variables on the stability of the human plasma proteome

Quality Control of Serum Albumin Depletion for Proteomic Analysis

Investigation of an albumin‐enriched fraction of human serum and its albuminome

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