Creation of a federated database of blood proteins: a powerful new tool for finding and characterizing biomarkers in serum

Ajambo

et al. 2017

Clin Proteom

Self Cite

The tryptic peptides from ice cold versus room temperature plasma were identified by C18 liquid chromatography and micro electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS). Samples collected on ice showed low levels of endogenous tryptic peptides compared to the same samples incubated at room temperature. Plasma on ice contained peptides from albumin, complement, and apolipoproteins and others that were observed by the X!TANDEM and SEQUEST algorithms. In contrast to ice cold samples, after incubation at room temperature, greater numbers of tryptic peptides from well characterized plasma proteins, and from cellular proteins were observed. A total of 583,927 precursor ions and MS/MS spectra were correlated to 94,669 best fit peptides that reduced to 22,287 correlations to the best accession within a gene symbol and to 7174 correlations to at least 510 gene symbols with ≥ 5 independent MS/MS correlations (peptide counts) that showed FDR q-values ranging from E−9 (i.e. FDR = 0.000000001) to E−227. A set of 528 gene symbols identified by X!TANDEM and SEQUEST including C4B showed ≥ fivefold variation between ice cold versus room temperature incubation. STRING analysis of the protein gene symbols observed from endogenous peptides in normal plasma revealed an extensive protein-interaction network of cellular factors associated with cell signalling and regulation, the formation of membrane bound organelles, cellular exosomes and exocytosis network proteins. Taken together the results indicated that a pool of cellular proteins, or protein complexes, in plasma are apparently not stable and degrade soon after incubation at room temperature.Electronic supplementary materialThe online version of this article (10.1186/s12014-017-9174-9) contains supplementary material, which is available to authorized users.

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The proteins cleaved by endogenous tryptic proteases in normal EDTA plasma by C18 collection of peptides for liquid chromatography micro electrospray ionization and tandem mass spectrometry

Dufresne

Ajambo

et al. 2017

Clin Proteom

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“…The peptides of blood fluids show good agreement on the detection of many unexpected cellular peptides by LC–ESI–MS/MS using both Qq-TOF and the sensitive ion trap [ 2 – 4 , 25 , 34 ]. The secretion or release of cellular proteins into extracellular space may result in the preferential cleavage of the cellular proteins upon exposure to circulating protease activities [ 35 ]. Here, the random and independent sampling of endogenous tryptic peptides were compared from plasma collected and incubated on ice versus the same plasma incubated at room temperature and showed many plasma proteins are degraded over time by tryptic proteases with about a twofold higher frequency of many tryptic peptides at room temperature compared to ice cold samples.…”

Section: Introductionmentioning

confidence: 99%

Random and independent sampling of endogenous tryptic peptides from normal human EDTA plasma by liquid chromatography micro electrospray ionization and tandem mass spectrometry

Dufresne

Ajambo

et al. 2017

Clin Proteom

Self Cite

BackgroundNormal human EDTA plasma samples were collected on ice, processed ice cold, and stored in a freezer at – 80 °C prior to experiments. Plasma test samples from the – 80 °C freezer were thawed on ice or intentionally warmed to room temperature.MethodsProtein content was measured by CBBR binding and the release of alcohol soluble amines by the Cd ninhydrin assay. Plasma peptides released over time were collected over C18 for random and independent sampling by liquid chromatography micro electrospray ionization and tandem mass spectrometry (LC–ESI–MS/MS) and correlated with X!TANDEM.ResultsFully tryptic peptides by X!TANDEM returned a similar set of proteins, but was more computationally efficient, than “no enzyme” correlations. Plasma samples maintained on ice, or ice with a cocktail of protease inhibitors, showed lower background amounts of plasma peptides compared to samples incubated at room temperature. Regression analysis indicated that warming plasma to room temperature, versus ice cold, resulted in a ~ twofold increase in the frequency of peptide identification over hours–days of incubation at room temperature. The type I error rate of the protein identification from the X!TANDEM algorithm combined was estimated to be low compared to a null model of computer generated random MS/MS spectra.ConclusionThe peptides of human plasma were identified and quantified with low error rates by random and independent sampling that revealed 1000s of peptides from hundreds of human plasma proteins from endogenous tryptic peptides.Electronic supplementary materialThe online version of this article (10.1186/s12014-017-9176-7) contains supplementary material, which is available to authorized users.

“…Illustration of the experimental setup to measure ELIMSA with absolute external PSA standards by the pyridoxamine (PA) production from the AP-SA probe alongside internal isotope dilution, internal one-point calibration, and external PA and 13 C PA standard curves. Panels: (A) internal 13 C isotope dilution curve for 12 NHP samples (left axis) that showed an average of ∼2.01 ± 0.0377 pmol of PA injected from each replicate well (right axis); (B) isocratic chromatogram showing the ELIMSA with an external PSA standard curve that was linear from 0.1 to 10 ng per well with 1.5 pmol 13 C internal standards, followed by 10 NHP samples with 1.5 pmol 13 C internal standards, and finally PA and 13 The amount of PA produced by the ELIMSA reactions was also estimated by the one-point calibration against the addition of 1.5 pmol of 13 Figure 3B, Table 1). There was good agreement between the independent internal isotope dilution curve and the one-point ratio estimate from a pooled normal sample that showed about 1.28 pmol of PA. On the same day, and on the same chromatogram as the one-point calibration experiment, we also measured PA production in terms of external 13 C PA and PA standard curves with the analysis of 0.1 μL analyzed from each 100 μL well.…”

Section: ■ Resultsmentioning

confidence: 99%

Pyridoxamine-5-phosphate Enzyme-Linked Immune Mass Spectrometric Assay Substrate for Linear Absolute Quantification of Alkaline Phosphatase to the Yoctomole Range Applied to Prostate Specific Antigen

Marshall

2014

Anal. Chem.

There is a need to measure proteins that are present in concentrations below the detection limits of existing colorimetric approaches with enzyme-linked immunoabsorbent assays (ELISA). The powerful enzyme alkaline phosphatase conjugated to the highly specific bacterial protein streptavidin binds to biotinylated macromolecules like proteins, antibodies, or other ligands and receptors with a high affinity. The binding of the biotinylated detection antibody, with resulting amplification of the signal by the catalytic production of reporter molecules, is key to the sensitivity of ELISA. The specificity and amplification of the signal by the enzyme alkaline phosphatase in ELISA together with the sensitivity of liquid chromatography electrospray ionization and mass spectrometry (LC-ESI-MS) to detect femtomole to picomole amounts of reporter molecules results in an ultrasensitive enzyme-linked immune mass spectrometric assay (ELIMSA). The novel ELIMSA substrate pyridoxamine-5-phosphate (PA5P) is cleaved by the enzyme alkaline phosphatase to yield the basic and hydrophilic product pyridoxamine (PA) that elutes rapidly with symmetrical peaks and a flat baseline. Pyridoxamine (PA) and (13)C PA were both observed to show a linear relationship between log ion intensity and quantity from picomole to femtomole amounts by liquid chromatography-electrospray ionization and mass spectrometry. Four independent methods, (i) internal (13)C isotope PA dilution curves, (ii) internal (13)C isotope one-point calibration, (iii) external PA standard curve, and (iv) external (13)C PA standard curve, all agreed within 1 digit in the same order of magnitude on the linear quantification of PA. Hence, a mass spectrometer can be used to robustly detect 526 ymol of the alkaline phosphatase streptavidin probe and accurately quantify zeptomole amounts of PSA against log linear absolute standard by micro electrospray on a simple ion trap.