Deep-ultraviolet laser ablation sampling for proteomic analysis of tissue

R.O. Lawal, L.T. Richardson, C. Dong, F. Donnarumma, T. Solouki, K.K. Murray, Deep-ultraviolet laser ablation sampling for proteomic analysis of tissue, Anal. Chim. Acta, 1184 (2021). doi: 10.1016/j.aca.2021.339021

Abstract

Deep-UV laser ablation sampling of tissue
Deep-ultraviolet laser ablation sampling for proteomic analysis of tissue

Deep-ultraviolet laser ablation with a pulsed 193 nm ArF excimer laser was used to remove localized regions from tissue sections from which proteins were extracted for spatially resolved proteomic analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). The ability to capture intact proteins by ablation at 193 nm wavelength was verified by matrix-assisted laser desorption ionization (MALDI) of the protein standard bovine serum albumin (BSA), which showed that BSA was ablated and captured without fragmentation. A Bradford assay of the ablated and captured proteins indicated 90% efficiency for transfer of the intact protein at a laser fluence of 3 kJ/m2. Rat brain tissue sections mounted on quartz microscope slides and ablated in transmission mode yielded 2 μg protein per mm2 as quantified by the Bradford assay. Tissue areas ranging from 0.06 mm2 to 1 mm2 were ablated and the ejected material was collected for proteomic analysis. Extracted proteins were digested and the resulting peptides were analyzed by LC-MS/MS. The proteins extracted from the ablated areas were identified and the average number of identified proteins ranged from 85 in the 0.06 mm2 area to 2400 in the 1 mm2 area of a 50 μm thick tissue. In comparison to infrared laser ablation of equivalent sampled areas, both the protein mass and number of proteins identified using DUV laser ablation sampling were approximately four times larger.

Multimodal Label‐Free Monitoring of Adipogenic Stem Cell Differentiation Using Endogenous Optical Biomarkers

N. Mehta, S. Shaik, A. Prasad, A. Chaichi, S.P. Sahu, Q. Liu, S.M.A. Hasan, E. Sheikh, F. Donnarumma, K.K. Murray, X. Fu, R. Devireddy, M.R. Gartia, Multimodal Label‐Free Monitoring of Adipogenic Stem Cell Differentiation Using Endogenous Optical Biomarkers, Adv. Funct. Mater., (2021) 2103955; doi: 10.1002/adfm.202103955

Abstract

Schematic overview of quantitative label-free imaging implemented at the single-cell level
Schematic overview of quantitative label-free imaging implemented at the single-cell level. a) Label-free monitoring of human adipose-derived stem cell (hASCs) differentiation to adipogenic stem cells using Raman spectroscopy; b) Metabolic imaging using second harmonic generation (SHG) of stem cells to detect differentiated stem cells from control cells; c) MALDI-mass spectrometry imaging of lipid in differentiated stem cells; d) Schematic arrangement showing the hyperspectral imaging (HSI) system. The HSI system captures the spatial and spectral signature at each pixel of the image to form the data cube.

Stem cell-based therapies carry significant promise for treating human diseases. However, clinical translation of stem cell transplants for effective treatment requires precise non-destructive evaluation of the purity of stem cells with high sensitivity (<0.001% of the number of cells). Here, a novel methodology using hyperspectral imaging (HSI) combined with spectral angle mapping-based machine learning analysis is reported to distinguish differentiating human adipose-derived stem cells (hASCs) from control stem cells. The spectral signature of adipogenesis generated by the HSI method enables identifying differentiated cells at single-cell resolution. The label-free HSI method is compared with the standard techniques such as Oil Red O staining, fluorescence microscopy, and qPCR that are routinely used to evaluate adipogenic differentiation of hASCs. HSI is successfully used to assess the abundance of adipocytes derived from transplanted cells in a transgenic mice model. Further, Raman microscopy and multiphoton-based metabolic imaging is performed to provide complementary information for the functional imaging of the hASCs. Finally, the HSI method is validated using matrix-assisted laser desorption/ionization-mass spectrometry imaging of the stem cells. The study presented here demonstrates that multimodal imaging methods enable label-free identification of stem cell differentiation with high spatial and chemical resolution.

Spatially resolved analysis of Pseudomonas aeruginosa biofilm proteomes measured by laser ablation sample transfer

A.C. Pulukkody, Y.P. Yung, F. Donnarumma, K.K. Murray, R.P. Carlson, L. Hanley, Spatially resolved analysis of Pseudomonas aeruginosa biofilm proteomes measured by laser ablation sample transfer, PLoS One, 16 (2021) e0250911, doi: 10.1371/journal.pone.0250911

Abstract

laser ablation sample transfer apparatus.
Schematic of laser ablation sample transfer (LAST) apparatus. A mid-IR laser beam is directed in transmission geometry through a glass slide to irradiate a biofilm. The spallated material is collected on a microcentrifuge tube for further processing.

Heterogeneity in the distribution of nutrients and oxygen gradients during biofilm growth
gives rise to changes in phenotype. There has been long term interest in identifying spatial
differences during biofilm development including clues that identify chemical heterogeneity.
Laser ablation sample transfer (LAST) allows site-specific sampling combined with label
free proteomics to distinguish radially and axially resolved proteomes for Pseudomonas aeruginosa
biofilms. Specifically, differential protein abundances on oxic vs. anoxic regions of a
biofilm were observed by combining LAST with bottom up proteomics. This study reveals a
more active metabolism in the anoxic region of the biofilm with respect to the oxic region for
this clinical strain of P. aeruginosa, despite this organism being considered an aerobe by
nature. Protein abundance data related to cellular acclimations to chemical gradients
include identification of glucose catabolizing proteins, high abundance of proteins from arginine
and polyamine metabolism, and proteins that could also support virulence and environmental
stress mediation in the anoxic region. Finally, the LAST methodology requires only a
few mm2 of biofilm area to identify hundreds of proteins.

Lasers for matrix-assisted laser desorption ionization

K.K. Murray, Lasers for matrix-assisted laser desorption ionization, J. Mass Spectrom., 56 (2021) e4664. https://doi.org/10.1002/jms.4664

Abstract

Lasers for MALDI
Lasers for MALDI Cover

Matrix-assisted laser desorption ionization (MALDI) was introduced 35 years ago and has advanced from a general method for producing intact ions from large biomolecules to wide use in applications ranging from bacteria identification to tissue imaging. MALDI was enabled by the development of high energy pulsed lasers that create ions from solid samples for analysis by mass spectrometry. The original lasers used for MALDI were ultraviolet fixed-wavelength nitrogen and Nd:YAG lasers, and a number of additional laser sources have been subsequently introduced with wavelengths ranging from the infrared to the ultraviolet and pulse widths from nanosecond to femtosecond. Wavelength tunable sources have been employed both in the IR and UV, and repetition rates have increased from tens of Hz to tens of kHz as MALDI has moved into mass spectrometry imaging. Dual-pulse configurations have been implemented with two lasers directed at the target or with a second laser creating ions in the plume of desorbed material. This review provides a brief history of the use of lasers for ionization in mass spectrometry and describes the various types of lasers and configurations used for MALDI.

In defense of the quasimolecular ion

K.K. Murray, In defense of the quasimolecular ion, J. Mass Spectrom., 56 (2021) e4700; doi: 10.1002/jms.4700.

Abstract

The term quasimolecular ion has been used to describe ions comprising a molecule and weakly bound positive or negative ion or an ion formed by the loss of a proton from a molecule. This term was used in mass spectrometry from the late 1960s after the development of chemical ionization but has been deprecated in recent terms recommendations due to what is perceived as its overly broad use. This letter argues that the term is well defined and has a long history of use in mass spectrometry and other fields and should be considered as a recommended term.

Mass Spectrometry Terms: Quasi-molecular ion