Open AccessEvaluation of Differential Peptide Loading on Tandem Mass Tag-Based Proteomic and Phosphoproteomic Data Quality
Author(s) -
James A. Sanford,
Yang Wang,
Joshua Hansen,
Marina Gritsenko,
Karl Weitz,
Tyler J Sagendorf,
Cristina E. Tog,
Vladislav Petyuk,
Weijun Qian,
Tao Liu,
Brian J. Druker,
Karin Rodland,
Paul Piehowski
Publication year - 2021
Publication title -
journal of the american society for mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.961
H-Index - 127
eISSN - 1879-1123
pISSN - 1044-0305
DOI - 10.1021/jasms.1c00169
Subject(s) - isobaric labeling , chemistry , peptide , tandem mass tag , proteome , multiplex , quantitative proteomics , tandem mass spectrometry , haematopoiesis , proteomics , phosphoproteomics , chromatography , computational biology , bioinformatics , biochemistry , mass spectrometry , biology , phosphorylation , stem cell , microbiology and biotechnology , protein phosphorylation , gene , protein kinase a
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.