Integration of Ion Mobility MSE after Fully Automated, Online, High-Resolution Liquid Extraction Surface Analysis Micro-Liquid Chromatography

Abstract

Direct analysis by mass spectrometry (imaging) has become increasingly deployed in preclinical and clinical research due to its rapid and accurate readouts. However, when it comes to biomarker discovery or histopathological diagnostics, more sensitive and in-depth profiling from localized areas is required. We developed a comprehensive, fully automated online platform for high-resolution liquid extraction surface analysis (HR-LESA) followed by micro-liquid chromatography (LC) separation and a data-independent acquisition strategy for untargeted and low abundant analyte identification directly from tissue sections. Applied to tissue sections of rat pituitary, the platform demonstrated improved spatial resolution, allowing sample areas as small as 400 μm to be studied, a major advantage over conventional LESA. The platform integrates an online buffer exchange and washing step for removal of salts and other endogenous contamination that originates from local tissue extraction. Our carry over-free platform showed high reproducibility, with an interextraction variability below 30%. Another strength of the platform is the additional selectivity provided by a postsampling gas-phase ion mobility separation. This allowed distinguishing coeluted isobaric compounds without requiring additional separation time. Furthermore, we identified untargeted and low-abundance analytes, including neuropeptides deriving from the pro-opiomelanocortin precursor protein and localized a specific area of the pituitary gland (i.e., adenohypophysis) known to secrete neuropeptides and other small metabolites related to development, growth, and metabolism. This platform can thus be applied for the in-depth study of small samples of complex tissues with histologic features of ∼400 μm or more, including potential neuropeptide markers involved in many diseases such as neurodegenerative diseases, obesity, bulimia, and anorexia nervosa.

Figure 1

Schematic representation of flow connections between the elements of the μLC system (in gray) and the automated sampler device (blue). Main features of the analytical platform are indicated in green. The route of the sample loop is shown in red. The 6-port valve is in the “sample loading” position. HR: high spatial resolution. LESA: liquid extraction surface analysis. BSM: binary solvent manager.

Figure 2

H&E stained images of the pituitary gland before (a) and after (b) HR-LESA extraction show an improvement in spatial resolution with HR-LESA (i.e., 400 μm area represented by a green dot in b) compared to conventional LESA (area shows the spot size of a typical LESA extraction (i.e., 1000 μm-diameter, red circle in b)). In (b), the arrow indicates the trace after an extraction with the capillary. Anatomical features of the pituitary gland containing the adenohypophysis and the neurohypophysis. Anatomical features: 1. anterior lobe; 2. posterior lobe; 3. intermediate lobe.

Figure 3

Sensitivity of LESA-MS versus HR-LESA-μLC-MS: The total mass spectrum (a and c) and a zoomed-in mass spectrum (b and d) of the adenohypophysis region of rat pituitary gland using LESA-MS (a and b) compared to HR-LESA-μLC-MS (c and d). The zoomed mass range displays a low-intensity peak corresponding to the protonated molecule of leucine enkephalin (marked with *), which is known to be present in this region. This is extracted from the ion chromatogram at a retention time at 16 min. The zoomed mass range from HR-LESA-μLC-MS shows the presence of leucine enkephalin coeluting with a triply charged species, which was not observed with LESA-MS.

Figure 4

Extraction of two coeluting compounds: the XIC (a) and corresponding mass spectra (b). The green XIC represents the chromatogram extracted from the monoisotopic peak of the triply charged species at m/z 555.65. The red XIC represents the chromatogram extracted from leucine enkephalin at m/z 556.28.

Figure 5

Ion mobilogram (a) of the extract from the anterior lobe of pituitary gland is depicted. The extracted ion mobility drift time spectrum (b) shows the separation of two coeluting compounds. The extracted mass spectra at drift time 3.8 (c) and 5.6 ms (d) show the separation of both compounds, indicated in red (leucine encephalin) and green (N-acetylated alpha-melanocyte stimulating hormone).