Microflow size exclusion chromatography to preserve micromolar affinity complexes and achieve subunit separations for native state mass spectrometry.

For high throughput native mass spectrometry (MS) protein characterization, it is advantageous to desalt and separate proteins by size exclusion chromatography (SEC). Sensitivity, resolution, and speed in these methods remain limited by standard SEC columns. Moreover, the efficient packing of small bore columns is notoriously difficult. SEC sensitivity is inherently limited because solutes are not focused into concentrated bands and low affinity native complexes may dissociate on column. Recent work evaluated the suitability of crosslinked gel media in small bore formats for online desalting. Here, small bore format online SEC for native MS studies is again investigated but with alternative materials. We systematically studied the utility of diol and hydroxy terminated polyethylene oxide (PEO) bonded 1.7 µm organosilica particles as packed into 1 mm ID stainless steel (SS) hardware and hardware treated with hydrophilic hybrid surface technology (h-HST). For the equivalent diol-bonded particle and hardware, UV limits of detection (LODs) were reduced 32 to 89% with a microflow separation (15 µL/min) on a 1 × 50 mm column as compared to a 4.6 × 150 mm high-flow separation (300 µL/min) at the same linear velocity. Run times were also shortened by 45%. A switch from SS to h-HST hardware led to a significant reduction in secondary interactions and a corresponding improvement in detection limits for trastuzumab, myoglobin, IgG and albumin for both UV and MS. Coupling of the small bore columns to multichannel microflow emitters resulted in 10 to 100-fold gains in MS sensitivity, depending on the analyte. MS LOD values were significantly reduced into the low attomole ranges. Columns were then evaluated for their effects on the preservation of complexes, including concanavalin A, in its apo and ligand-bound states, and three therapeutically relevant noncovalent systems previously undetected on large column formats. The results suggest that the detection of large complexes by SEC is not just a function of sensitivity but is directly affected by chemical secondary interactions. The ability to detect 0.1 to 1 MDa complexes, with between 1 and 40 micromolar dissociation constants, represents a critical advancement for high-throughput native MS workflows as applied to the analysis of therapeutics.

Delineation of disease phenotypes associated with esophageal adenocarcinoma by MALDI-IMS-MS analysis of serum N-linked glycans.

N-Linked glycans, extracted from patient sera and healthy control individuals, are analyzed by Matrix-assisted laser desorption ionization (MALDI) in combination with ion mobility spectrometry (IMS), mass spectrometry (MS) and pattern recognition methods. MALDI-IMS-MS data were collected in duplicate for 58 serum samples obtained from individuals diagnosed with Barrett's esophagus (BE, 14 patients), high-grade dysplasia (HGD, 7 patients), esophageal adenocarcinoma (EAC, 20 patients) and disease-free control (NC, 17 individuals). A combined mobility distribution of 9 N-linked glycans is established for 90 MALDI-IMS-MS spectra (training set) and analyzed using a genetic algorithm for feature selection and classification. Two models for phenotype delineation are subsequently developed and as a result, the four phenotypes (BE, HGD, EAC and NC) are unequivocally differentiated. Next, the two models are tested against 26 blind measurements. Interestingly, these models allowed for the correct phenotype prediction of as many as 20 blinds. Although applied to a limited number of blind samples, this methodology appears promising as a means of discovering molecules from serum that may have capabilities as markers of disease.

Chemical mapping of the colorectal cancer microenvironment via MALDI imaging mass spectrometry (MALDI-MSI) reveals novel cancer-associated field effects.

Matrix-assisted laser desorption ionisation imaging mass spectrometry (MALDI-MSI) is a rapidly advancing technique for intact tissue analysis that allows simultaneous localisation and quantification of biomolecules in different histological regions of interest. This approach can potentially offer novel insights into tumour microenvironmental (TME) biochemistry. In this study we employed MALDI-MSI to evaluate fresh frozen sections of colorectal cancer (CRC) tissue and adjacent healthy mucosa obtained from 12 consenting patients undergoing surgery for confirmed CRC. Specifically, we sought to address three objectives: (1) To identify biochemical differences between different morphological regions within the CRC TME; (2) To characterise the biochemical differences between cancerous and healthy colorectal tissue using MALDI-MSI; (3) To determine whether MALDI-MSI profiling of tumour-adjacent tissue can identify novel metabolic 'field effects' associated with cancer. Our results demonstrate that CRC tissue harbours characteristic phospholipid signatures compared with healthy tissue and additionally, different tissue regions within the CRC TME reveal distinct biochemical profiles. Furthermore we observed biochemical differences between tumour-adjacent and tumour-remote healthy mucosa. We have referred to this 'field effect', exhibited by the tumour locale, as cancer-adjacent metaboplasia (CAM) and this finding builds on the established concept of field cancerisation.