TY - JOUR
T1 - Evaluation of Differential Peptide Loading on Tandem Mass Tag-Based Proteomic and Phosphoproteomic Data Quality
AU - Sanford, James A.
AU - Wang, Yang
AU - Hansen, Joshua R.
AU - Gritsenko, Marina A.
AU - Weitz, Karl K.
AU - Sagendorf, Tyler J.
AU - Tognon, Cristina E.
AU - Petyuk, Vladislav A.
AU - Qian, Wei Jun
AU - Liu, Tao
AU - Druker, Brian J.
AU - Rodland, Karin D.
AU - Piehowski, Paul D.
N1 - Funding Information:
Parts of this research were performed using EMSL, a national scientific user facility sponsored by the Department of Energy’s Office of Biological and Environmental Research and located at PNNL.
Funding Information:
This work was supported by The Proteogenomic Translational Research Center for Clinical Proteomics Tumor Analysis Consortium, U01 CA214116, from NIH NCI to B.J.D. and K.D.R., U24CA210955 from NIH NCI to T.L., and R01 DK122160 from NIH NIDDK to W.-J.Q.
Funding Information:
Parts of this research were performed using EMSL, a national scientific user facility sponsored by the Department of Energy?s Office of Biological and Environmental Research and located at PNNL.
Publisher Copyright:
© 2021 Battelle Memorial Institute. Published by American Chemical Society
PY - 2022/1/5
Y1 - 2022/1/5
N2 - Global and phosphoproteome profiling has demonstrated great utility for the analysis of clinical specimens. One barrier to the broad clinical application of proteomic profiling is the large amount of biological material required, particularly for phosphoproteomics─currently on the order of 25 mg wet tissue weight. For hematopoietic cancers such as acute myeloid leukemia (AML), the sample requirement is ≥10 million peripheral blood mononuclear cells (PBMCs). Across large study cohorts, this requirement will exceed what is obtainable for many individual patients/time points. For this reason, we were interested in the impact of differential peptide loading across multiplex channels on proteomic data quality. To achieve this, we tested a range of channel loading amounts (approximately the material obtainable from 5E5, 1E6, 2.5E6, 5E6, and 1E7 AML patient cells) to assess proteome coverage, quantification precision, and peptide/phosphopeptide detection in experiments utilizing isobaric tandem mass tag (TMT) labeling. As expected, fewer missing values were observed in TMT channels with higher peptide loading amounts compared to lower loadings. Moreover, channels with a lower loading have greater quantitative variability than channels with higher loadings. A statistical analysis showed that decreased loading amounts result in an increase in the type I error rate. We then examined the impact of differential loading on the detection of known differences between distinct AML cell lines. Similar patterns of increased data missingness and higher quantitative variability were observed as loading was decreased resulting in fewer statistical differences; however, we found good agreement in features identified as differential, demonstrating the value of this approach.
AB - Global and phosphoproteome profiling has demonstrated great utility for the analysis of clinical specimens. One barrier to the broad clinical application of proteomic profiling is the large amount of biological material required, particularly for phosphoproteomics─currently on the order of 25 mg wet tissue weight. For hematopoietic cancers such as acute myeloid leukemia (AML), the sample requirement is ≥10 million peripheral blood mononuclear cells (PBMCs). Across large study cohorts, this requirement will exceed what is obtainable for many individual patients/time points. For this reason, we were interested in the impact of differential peptide loading across multiplex channels on proteomic data quality. To achieve this, we tested a range of channel loading amounts (approximately the material obtainable from 5E5, 1E6, 2.5E6, 5E6, and 1E7 AML patient cells) to assess proteome coverage, quantification precision, and peptide/phosphopeptide detection in experiments utilizing isobaric tandem mass tag (TMT) labeling. As expected, fewer missing values were observed in TMT channels with higher peptide loading amounts compared to lower loadings. Moreover, channels with a lower loading have greater quantitative variability than channels with higher loadings. A statistical analysis showed that decreased loading amounts result in an increase in the type I error rate. We then examined the impact of differential loading on the detection of known differences between distinct AML cell lines. Similar patterns of increased data missingness and higher quantitative variability were observed as loading was decreased resulting in fewer statistical differences; however, we found good agreement in features identified as differential, demonstrating the value of this approach.
KW - TMT
KW - acute myeloid leukemia
KW - clinical proteomics
KW - differential loading
KW - isobaric labeling
KW - phosphoproteomics
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U2 - 10.1021/jasms.1c00169
DO - 10.1021/jasms.1c00169
M3 - Article
C2 - 34813325
AN - SCOPUS:85120381254
SN - 1044-0305
VL - 33
SP - 17
EP - 30
JO - Journal of the American Society for Mass Spectrometry
JF - Journal of the American Society for Mass Spectrometry
IS - 1
ER -