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

Jaimie Dufresne,Peter Bowden,Ammara Ferwa,Juliet Ajambo,Angelique Florentinus-Mefailoski,John Marshall

doi:10.1186/s12014-017-9174-9

Jaimie Dufresne, Peter Bowden + Show 4 more

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https://doi.org/10.1186/s12014-017-9174-9

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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.

Highlights

Many of the well-known proteins of human blood [1, 2] are cleaved by endogenous tryptic endopeptidases to release fully tryptic peptides that may be identified by collection over C18 followed by liquid chromatography, electrospray ionization and tandem mass spectrometry (LC–ESI–MS/MS) with a Paul ion trap or Qq-TOF [3,4,5].Exopeptidases are active in blood fluid that give rise to non-tryptic peptides that are computationally challenging to identify [6,7,8]
In addition to TTN, the analysis of source noise from blank runs indicated that ZNF503, VWCE, API5, TMEM199, SYT7, TPSG1, USP9X, DACT3, SUGP2, CXorf31, MYO3A, KLRC4, CYCS, RGP1, SYN1, DSPP, IMMP1L CAMK1, ASPM, AHNAK, among others were correlated by SEQUEST from noise spectra collected from LC–ESI–MS/MS gradient runs with blank samples [28, 29]
The Type I error from correlating experimental MS/MS spectra to those predicted for human tryptic peptides was limited by X!TANDEM or by taking the ratio of room temperature versus preserved samples that was supported by the comparison to the null model of random peptides and the non-random distribution across Gene Ontology (GO) terms

Summary

Introduction

Many of the well-known proteins of human blood [1, 2] are cleaved by endogenous tryptic endopeptidases to release fully tryptic peptides that may be identified by collection over C18 followed by liquid chromatography, electrospray ionization and tandem mass spectrometry (LC–ESI–MS/MS) with a Paul ion trap or Qq-TOF [3,4,5].Exopeptidases are active in blood fluid that give rise to non-tryptic peptides that are computationally challenging to identify [6,7,8]. Many of the well-known proteins of human blood [1, 2] are cleaved by endogenous tryptic endopeptidases to release fully tryptic peptides that may be identified by collection over C18 followed by liquid chromatography, electrospray ionization and tandem mass spectrometry (LC–ESI–MS/MS) with a Paul ion trap or Qq-TOF [3,4,5]. Incubating blood fluid samples at room temperature releases tryptic peptides that have been confirmed by high mass accuracy measurements of the parent peptide masses and the use of tryptic protease inhibitors [5, 8]. The endogenous peptides of normal plasma may contain fragments of commonly known blood proteins released from tissues, cells and organs that might be cleaved by proteases ex vivo [3,4,5]. Sampling variation was addressed by collecting EDTA plasma directly onto ice and purposefully degrading samples at room temperature under controlled conditions

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