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Evaluation of decontamination methods for palaeoproteomic analysis of Pleistocene skeletal proteomes

ABSTRACT: Palaeoproteomic analysis of skeletal elements is increasingly used to study topics such as human evolution. However, many of the skeletal elements that are analysed have been stored and handled over long time periods, and have therefore accumulated contaminating proteins, which may complicate analysis of the endogenous ancient proteome. In this study, we evaluate five commonly used approaches for decontamination in palaeoproteomics, using a Pleistocene Equus sp. bone that was artificially contaminated with modern dog saliva and fur. First, we show that contamination affects reconstruction of the ancient proteome, even when the potential for contamination is not taken into account. Thereafter, our results show that although most decontamination methods reduce contamination, only a brief bleach wash seemingly returns the proteome to its original composition. None of the decontamination approaches are found to damage the endogenous proteome. Decontamination may, however, significantly improve recovery of low-abundance proteins.

INSTRUMENT(S):

ORGANISM(S): Equus Caballus (horse) Canis Familiaris (dog) (canis Lupus Familiaris)

TISSUE(S): Hair, Bone, Saliva

SUBMITTER: Zandra Fagernäs

LAB HEAD: Frido Welker

PROVIDER: PXD050393 | Pride | 2025-04-24

REPOSITORIES: Pride

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			Action	DRS
	20230525_EXPL8_nLC11_GT_collab_77min_DDA_Blank_P0408.raw	Raw
	20230525_EXPL8_nLC11_GT_collab_77min_DDA_P0387.raw	Raw
	20230525_EXPL8_nLC11_GT_collab_77min_DDA_P0388.raw	Raw
	20230525_EXPL8_nLC11_GT_collab_77min_DDA_P0389_20230528201553.raw	Raw
	20230525_EXPL8_nLC11_GT_collab_77min_DDA_P0390.raw	Raw

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Project description:Background. Flow cytometry (FCM) identifies leukemic blasts in cerebrospinal fluid (CSF) in approximately 30% of children with acute lymphoblastic leukemia (ALL), whereas conventional cytospin preparations frequently miss such involvement. <br>Procedure. To explore biological properties of leukemic cells that migrate into central nervous system (CNS), we compared gene expression profiles of leukemic cells from 26 patients with CNS involvement and 138 patients without CNS leukemia as determined by FCM and/or cytospin preparations. Results were validated by real-time quantitative PCR (qPCR). <br>Results. We found the cysteine endopeptidase legumain (LGMN) that plays a role in hydrolysis of proteins and small molecules to be significantly differentially expressed between FCM+ and FCM- B-cell precursor (BCP) ALL patients. When LGMN expression was validated by qPCR it stayed differentially expressed between FCM+ and FCM- BCP-ALL patients (P=0.006), and also between FCM+ and cytospin-negative BCP-ALL patients (P=0.01). In multivariate regression analysis including sex, age and WBC, LGMN expression levels above population median were associated with an odds ratio of 9.1 for CNS involvement as determined by FCM compared with expression less than the median (95% CI, 1.26 to 65.50; P=0.03). Conclusions. The use of more sensitive methods for detecting and quantifying CNS involvement, such as FCM, may facilitate detection of markers of leukemia biology, such as LGMN, which could have therapeutical implications for future CNS-directed therapy.<br>FCM+: Patients had leukemic cells in the cerebrospinal fluid (CSF) detected by flow cytometry and no blood contamination. FCM-: Patients had no leukemic cells in the CSF detected by flow cytometry with or without blood contamination.<br>CNS1: No leukemic cells in CSF detected by cytospin examination with or without blood contamination and no other clinical signs of central nervous system (CNS) leukaemia. CNS2: CSF leukocyte count <5 x106/ÂµL with blasts on cytospin examination and no blood contamination. CNS3: CSF leukocyte count >5 x106/ÂµL with blasts on cytospin examination and no blood contamination, and/or cranial nerve palsy, and/or CNS leukaemia by neuroimaging.

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Evaluation of decontamination methods for palaeoproteomic analysis of Pleistocene skeletal proteomes

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