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Previous meta-analysis indicated that plasma or serum proteome groups using various experimental conditions detected different peptides from the same plasma proteins, which is strong evidence for the veracity of blood fluid LC-ESI-MS/MS but also evidences that the trypsin digestion step is a key source of variation in plasma proteomics. Agreement between different digestion conditions and MS/MS algorithms may serve as an independent confirmation of the validity of the LC-ESI-MS/MS analysis of plasma peptides. Plasma contains a high percentage of albumin held together by multiple disulfide bonds; hence, reduction and/or alkylation may greatly enhance the digestion efficiency of albumin. Plasma proteins were precipitated in 90% acetonitrile, collected over quaternary amine resin, and eluted in NaCl prior to digestion treatments. To determine the effect of trypsin digestion methods, the plasma proteins were digested in 600 mM urea and 5% acetonitrile with trypsin alone, or reduced with 2 mM DTT followed by trypsin, or DTT followed by 15 mM iodoacetamide and then trypsin. The resulting peptides were analyzed by LC-ESI-MS/MS with a linear quadrupole ion trap (LIT). The MS/MS spectra were directly fit to peptides by the X!TANDEM and SEQUEST algorithms. Blank noise injections served as the analytical control, and 30 million random MS/MS served as the statistical control. Digesting human plasma with DTT reduction, or reduction and alkylation, resulted in a dramatic increase in the number and observation frequency of albumin peptides. In contrast, digestion with trypsin alone suppressed the observation of albumin, and instead, many low abundance plasma and cellular proteins showed higher observation frequency. Digestion with trypsin alone increased the observation frequency of APOC1, ACAN, ATRN, CPB2, GP2, GPX3, HBA1, PAPD5, PKD1, and many cellular proteins. After correction against noise and random controls, SEQUEST showed good agreement with the true positive plasma proteins identified by X!TANDEM and resulted in an R -squared of 0.5238 with an F -statistic of 10,930 on 9,935 protein gene symbols with a p -value < 2.2e-16. Digestion of plasma with trypsin alone avoids the complete digestion of albumin and permits the enhanced detection of some other cellular proteins from plasma. Different digestion approaches were complimentary and together resulted in a more comprehensive plasma proteome. The protein FDR q -values, the modest effect of background and Monte Carlo correction, and the significant STRING analysis were all consistent with the high fidelity of the rigorous X!TANDEM algorithm. In contrast, SEQUEST required significant correction against noise and statistical controls and selection of high cross correlation (XCorr) scores to show good agreement with X!TANDEM. There was qualitative and quantitative agreement between plasma proteins digested without alkylation from the orbital ion trap (OIT) versus the LIT instrument that showed highly significant regression against the X!TANDEM OIT monoisotopic results, those from heavy isotopes and other masses from X!TANDEM, and with those from MaxQuant. There was significant qualitative and quantitative agreement between the complementary digestion conditions consistent with the good fidelity of plasma analysis by LC-ESI-MS/MS with a sensitive linear ion trap., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

The naturally occurring peptides and digested proteins of fetal versus adult bovine serum were compared by LC-ESI-MS/MS after correction against noise from blank injections and random MS/MS spectra as statistical controls. Serum peptides were extracted by differential precipitation with mixtures of acetonitrile and water. Serum proteins were separated by partition chromatography over quaternary amine resin followed by tryptic digestion. The rigorous X!TANDEM goodness of fit algorithm that has a low error rate as demonstrated by low FDR q-values (q ≤ 0.01) showed qualitative and quantitative agreement with the SEQUEST cross correlation algorithm on 12,052 protein gene symbols. Tryptic digestion provided a quantitative identification of the serum proteins where observation frequency reflected known high abundance. In contrast, the naturally occurring peptides reflected the cleavage of common serum proteins such as C4A, C3, FGB, HPX, A2M but also proteins in lower concentration such as F13A1, IK, collagens and protocadherins. Proteins associated with cellular growth and development such as actins (ACT), ribosomal proteins like Ribosomal protein S6 (RPS6), synthetic enzymes and extracellular matrix factors were enriched in fetal calf serum. In contrast to the large literature from cord blood, IgG light chains were absent from fetal serum as observed by LC-ESI-MS/MS and confirmed by ELISA., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)