posted on 2021-11-23, 18:16authored byJames
A. Sanford, Yang Wang, Joshua R. Hansen, Marina A. Gritsenko, Karl K. Weitz, Tyler J. Sagendorf, Cristina E. Tognon, Vladislav A. Petyuk, Wei-Jun Qian, Tao Liu, Brian J. Druker, Karin D. Rodland, Paul D. Piehowski
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 phosphoproteomicscurrently
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.