Sensitive, robust and automated protein analysis of cell differentiation and of primary human blood cells by intact cell MALDI mass spectrometry biotyping

Abstract

Intact cell mass spectrometry biotyping, a collection of methods for classification of cells based on mass spectrometric fingerprints, is an established method in clinical and environmental microbiology. It has recently also been applied to the investigation of mammalian cells including primary blood cells and cultured cells. However, few automated procedures suitable for higher throughput and little analytical standardization of mammalian biotyping approaches have been reported so far. Here, we present a novel automated method that robustly classifies as few as 250 cells per spot. Automatically acquired cell fingerprints from cultured and primary cells show high technical (R > 0.95) and biological reproducibility (R = 0.83-0.96), with a median peak variance below 12 %. Ion suppression is shown to be a major concern at higher cell numbers and needs to be carefully monitored. We demonstrate that intact cell mass spectrometric signatures of different cell lines start to resemble each other at higher trifluoroacetic acid (TFA) concentrations and that therefore low concentrations of TFA in the matrix solution are preferred. We show that in vitro differentiation of HL-60 cells into a neutrophil-like phenotype can be rapidly and robustly monitored. We utilize the method for global analysis of person-to-person differences in mass spectral signatures of intact polymorphonuclear neutrophils and monocytes obtained from healthy volunteers. Our data suggest that automated MALDI mass spectrometry cell biotyping could be a useful complementary approach in clinical cell analysis.

Rapid high-throughput characterisation, classification and selection of recombinant mammalian cell line phenotypes using intact cell MALDI-ToF mass spectrometry fingerprinting and PLS-DA modelling

“…Despite the sensitivity, success and simplicity of MALDI-ToF coupled with appropriate data analysis for characterising bacteria, this approach has not been routinely utilised for the analysis of mammalian cells. Whilst there have been several reports of applying intact cell MALDI-ToF for the analysis of different mammalian cell lines and processes (Hanrieder et al, 2011;Karger et al, 2010;Marvin-Guy et al, 2008;Munteanu et al, 2012;Zhang et al, 2006) and to the analysis of CHO cells (Feng et al, 2011;Feng et al, 2010), none of these studies has attempted to use such an approach to predict across scale (e.g. use data early in the cell line construction process to predict a cells productivity at larger scale).…”

Profiling embryonic stem cell differentiation by MALDI TOF mass spectrometry: development of a reproducible and robust sample preparation workflow

“…In our experience, harsh lysing conditions resulted in spectra that were less distinguishable, which has also been observed by lysing with increasing acidity. 37 It was indicated before that freeze-thawing of cell pellets prior to MALDI TOF MS analysis may have beneficial effects with respect to the number of features identified and overall spectral intensity. 33,46 This is likely due to the freeze/thaw cycle permeating the cell membrane, thus allowing the cytoplasmic contents of the cells to become exposed and more easily ionised.…”

“…Characterising the protein signatures of mammalian cells by MALDI-MS is less common when compared with lipid analysis; however it has been used for phenotypic screening of human cancer cell lines, 31,32 identification of cells within a co-culture 33,34 or tissues 35 and detection of transient changes within a specific cell type, such as immune cells. [36][37][38][39] However, many of these studies list dramatically different experimental procedures with several being adapted from existing biotyping protocols. The huge range of experimental parameters could therefore be problematic for translation of published assays to the pharmaceutical industry.…”

Whole-cell MALDI-TOF Mass Spectrometry is an Accurate and Rapid Method to Analyze Different Modes of Macrophage Activation

“…The cell concentration may be a limit for specific clinical applications such as the analysis of needle biopsies or cells harvested from broncho-alveolar lavages. A recent article described an automated approach of wholecell MALDI-TOF analysis that allowed the robust classification of samples with as few as 250 cells on each spot 11 . A proof of concept of the clinical application of this technique to the diagnosis of oral cancer has also been recently published 15 .…”

“…Many applications may be derived from this technique: (1) identify the main cell types from a mixed sample [9][10][11] ; (2) assess the viability of cell cultures over time (including quality control industrial applications) 12 ; (3) monitor activation states of a single cell type 13 ; (4) assess the malignant transformation of a clinical sample 14,15 .…”