Rationale Histone post‐translational modifications (PTMs) play key roles in regulating eukaryotic gene expression. Mass spectrometry (MS) has emerged as a powerful method to characterize and quantify histone PTMs as it allows unbiased identification and quantification of multiple histone PTMs including combinations of the modifications present. Methods In this study we compared a range of data‐acquisition methods for the identification and quantification of the histone PTMs using a Q Exactive HF Orbitrap. We compared three different data‐dependent analysis (DDA) methods with MS2 resolutions of 120K, 60K, 30K. We also compared a range of data‐independent analysis (DIA) methods using MS2 isolation windows of 20 m/z and DIAvw to identify and quantify histone PTMs in Chinese hamster ovary (CHO) cells. Results The increased number of MS2 scans afforded by the lower resolution methods resulted in a higher number of queries, peptide sequence matches (PSMs) and a higher number of peptide proteoforms identified with a Mascot Ion score greater than 46. No difference in the proportion of peptide proteoforms with Delta scores >17 was observed. Lower coefficients of variation (CVs) were obtained in the DIA MS1 60 K MS2 30 K 20 m/z isolation windows compared with the other data‐acquisition methods. Conclusions We observed that DIA which offers advantages in flexibility and identification of isobaric peptide proteoforms performs as well as DDA in the analysis of histone PTMs. We were able to identify 71 modified histone peptides for histone H3 and H4 and quantified 64 across each of the different acquisition methods.
The U.S. Food and Drug Administration Food Code suggests that holding fresh-cut produce at < 5°C will limit growth of pathogenic microorganisms. Here, we determined whether cucumber, onion, pepper, mango, and tomato supported growth of Listeria monocytogenes (LM), Shiga toxin-producing Escherichia coli (STEC), and Salmonella enterica (SALM) at 5, 10, and 22°C.Produce was surface-pasteurized, diced, inoculated with single-pathogen cocktails, and incubated. Survivors were then enumerated with change in population (Δ-log CFU per gram) determined over time. Mango did not support pathogen growth at 5 or 10°C, but SALM and STEC exhibited significant (P < 0.05) growth on mango at 22°C (2.85 and 1.41 Δ-log CFU/g, respectively). At 5°C, significant (P < 0.05) growth was seen on cucumber inoculated with SALM and LM; onion and pepper inoculated with LM; and tomato inoculated with STEC. At 10°C, freshcut cucumber, onion, and pepper supported significant (P < 0.05) increases in SALM, STEC, and LM, along with SALM on tomato; Δ-log ranged from 3.37 (onion, LM) to 5.40 CFU/g (pepper, SALM). Growth of pathogens was not significantly different (P < 0.05) at 10 and 22°C for SALM or STEC inoculated onto onion, pepper, cucumber, or tomato. Results suggest that holding fresh-cut produce at or near refrigeration temperatures (5 or 10°C) may not control risk of pathogen growth.
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