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

Abstract

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.

Preanalytical sample handling recommendations for Alzheimer's disease plasma biomarkers

“…Several prior studies have demonstrated that delayed centrifugation of whole blood impacts the analysis of biochemical components in plasma and serum samples [30]. Quantification of blood‐based protein biomarkers may be severely impaired due to, for example, degradation by multiple proteolytic enzymes in the blood, or in the plasma or serum present in supernatant after complete blood coagulation [31–33]. Moreover, the quantitative measurement of AD biomarkers might be affected by aggregation‐dependent depletion of the monomeric forms of Aβ(1‐40) and Aβ(1‐42).…”

The plasma peptides of ovarian cancer

“…Blood fluid contains a net weak tryptic activity [57] that may cleave endogenous peptides in vivo (peptidome) and endogenous proteolytic activities generate high levels of some of these same peptides ex vivo (degradome) [58, 59] where these two pools show some overlap [2]. The frequency and/or intensity of peptide observations increased in samples incubated at room temperature compared to ice cold samples that shared some peptides and proteins [1–3, 5, 24]. The increased frequency and average precursor intensity values of cellular proteins across the clinical samples compared to the ice cold controls indicates the some of the peptides and or proteins observed were released from cells, or degraded by proteases released or activated, ex vivo.…”

The plasma peptidome

“…We previously showed that plasma from blood collected into EDTA tubes on ice is stable when freeze dried with low peptide frequency and intensity but starts to degrade when dissolved at room temperature [ 27 , 29 , 47 ]. The frequency and/or intensity of peptide or protein observations increased in samples incubated at room temperature compared to ice cold samples and the two pools shared some peptides and proteins [ 16 , 27 , 28 , 38 , 47 ]. Differences in the frequency of observation and average precursor intensity values of specific cellular proteins like RAB1 or DENDD5A across the clinical samples compared to the ice cold controls indicates the at least some of the peptides and or proteins observed have been released from cells, or degraded by proteases released or activated, ex vivo.…”

“…Exogenous tryptic digestion of blood fluids results in the highly redundant analysis of albumin, apolipoproteins, immunoglobulins [ 14 , 15 ], and other well-known blood proteins [ 1 , 2 ]. In contrast, examination of endogenous peptides shows a greater representation of apparently cellular proteins [ 16 , 17 ]. The agreement on the identified proteins of human blood fluids from MS/MS spectra between “Fully Tryptic” peptides that are constrained to end in R or K [ 8 , 15 ] versus the “No Enzyme” peptides that are free to end with any of the 20 amino acids [ 10 , 11 ] is powerful evidence for the veracity of LC–ESI–MS/MS of tryptic peptides [ 14 , 18 , 19 ].…”

“…Organic extraction may have some advantage to detect cellular proteins compared to the redundant identification of canonical circulating proteins frequently observed from depletion chromatography [ 10 ], ultrafiltration [ 11 ], or partition chromatography [ 8 , 15 ] of blood proteins followed by trypsin digestion. Precipitating the blood peptides for organic/water extraction has resulted in the identification of cellular proteins and regulatory molecules growth factors [ 16 , 17 , 36 , 37 ]. It will be necessary to identify, quantify and compute the statistical distributions of the endogenous tryptic peptides cleaved from the proteins ex vivo in blood plasma compared to ice cold controls to understand and compare treatment versus pre-analytical variation in different clinical populations and controls.…”