Robustness and Ruggedness of Isoelectric Focusing and Superficially Porous Liquid Chromatography with Fourier Transform Mass Spectrometry.

An investigation of a multidimensional proteomics workflow composed of off-gel isoelectric focusing (IEF) and superficially porous liquid chromatography (SPLC) with Fourier transform mass spectrometry (FTMS) was completed in order to assess various figures of merit associated with intact protein measurements. Triplicate analysis performed at both high and low FTMS resolutions on the E. coli proteome resulted in ∼900 redundant proteoforms from 3 to 95 kDa. Normalization of the chromatographic axis to identified proteoforms enabled reproducible physicochemical property measurements between proteome replicates with inter-replicate variances of ±3 ppm mass error for proteoforms <30 kDa, ±1.1 Da for proteins >30 kDa, ±12 s retention time error, and ±0.21 pI units. The results for E. coli and standard proteins revealed a correlation between pI precision and proteoform abundance with species detected in multiple IEF fractions exhibiting pI precisions less than the theoretical resolution of the off-gel system (±0.05 vs ±0.17, respectively). Evaluation of differentially modified proteoforms of standard proteins revealed that high sample loads (100s µgrams) change the IEF pH gradient profile, leading to sample broadening that facilitates resolution of charged post-translational modifications (e.g., phosphorylation, sialylation). Despite the impact of sample load on IEF resolution, results on standard proteins measured directly or after being spiked into E. coli demonstrated that the reproducibility of the workflow permitted recombination of the MS signal across IEF fractions in a manner supporting the evaluation of three label-free quantitation metrics for intact protein studies (proteoforms, proteoform ratios, and protein) over 102-103 sample amount with low femtomole detection limits.

Proteoform analysis of lipocalin-type prostaglandin D-synthase from human cerebrospinal fluid by isoelectric focusing and superficially porous liquid chromatography with Fourier transform mass spectrometry.

Lipocalin-type prostaglandin D-synthase (L-PGDS) in cerebrospinal fluid contributes to the maturation and maintenance of the CNS. L-PGDS PTMs may contribute to pathobiology of different CNS diseases, but methods to monitor its proteoforms are limited. Herein, we combined off-gel IEF and superficially porous LC (SPLC) with Fourier transform MS to characterize common cerebrospinal fluid L-PGDS proteoforms. Across 3D physiochemical space (pI, hydrophobicity, and mass), 217 putative proteoforms were observed from 21 to 24 kDa and pI 5-10. Glycoprotein accurate mass information, combined with MS/MS analysis of peptides generated from 2D-fractionated proteoforms, enabled the putative assignment of 208 proteoforms with varied PTM positional occupants. Fifteen structurally related N-glycans at N29 and N56 were observed, with different N-glycan compositional variants being preferred on each amino acid. We also observed that sialic acid content was a major factor for pI shifts between L-PGDS proteoforms. Other putative PTMs characterized include a core-1 HexHexNAc-O-glycan at S7, acetylation at K16 and K138, sulfonation at S41 and T142, and dioxidation at C43 and C145. The IEF-SPLC-MS platform presented provides 30-40× improved peak capacity versus conventional 2DE and shows potential for repeatable proteoform analysis of surrogate PTM-based biomarkers.

Top-down mass spectrometry on tissue extracts and biofluids with isoelectric focusing and superficially porous silica liquid chromatography.

Top-down mass spectrometry (MS) has emerged as a powerful complement to peptide-based proteomics. Despite advancements, the field has had limited application to clinical proteomics investigations due to the complexity and poor dynamic range of chromatography used to separate intact proteins from tissue and biofluids. To address these limitations, we developed a two-dimensional (2D) chromatography platform that includes isoelectric focusing (IEF) through immobilized pH gradient and superficially porous liquid chromatography (SPLC). Analysis of standard proteins demonstrates compatibility of IEF-SPLC processing and high resolving-power MS analysis with results showing ~7.0 femtomole detection limits and linear spectral response for proteins fractionated over ~4 log sample loads. For proteins from heart myofibrils and cerebrospinal fluid (CSF), compared to one-dimensional SPLC-MS, the 2D IEF-SPLC-MS platform resulted in a 5-6× increase in the number of unique monoisotopic masses observed <30 kDa and an ~4× improved mass range enabling the observation of proteins >200 kDa. In the heart myofibrils, common protein proteoforms observed were associated with phosphorylation of contractile proteins with results showing that quantitative evaluation of their PTM stoichiometry was possible despite differentially modified forms being fractionated into separate pI compartments. In CSF, diverse protein mutations and PTM classes were also observed, including differentially glycosylated protein forms separated to different pI. Results also demonstrate that by the generation of IEF-SPLC protein libraries by fraction collection, the platform enables prospective protein identification and proteoform analysis investigations by complementary top-down and bottom-up strategies. Overall, the 2D platform presented may provide the speed, dynamic range, and detection limits necessary for routine characterization of proteoform-based biomarkers from biofluids and tissues.

Surface preparation strategies for improved parallelization and reproducible MALDI-TOF MS ligand binding assays.

Immunoassays are employed in academia and the healthcare and biotech industries for high-throughput, quantitative screens of biomolecules. We have developed monolayer-based immunoassays for MALDI-TOF MS. To improve parallelization, we adapted the workflow to photolithography-generated arrays. Our work shows Parylene-C coatings provide excellent "solvent pinning" for reagents and biofluids, enabling sensitive MS detection of immobilized components. With a unique MALDI-matrix crystallization technique we show routine interassay RSD <10% at picomolar concentrations and highlight platform compatibility for relative and label-free quantitation applications. Parylene-arrays provide high sample densities and promise screening throughputs exceeding 1000 samples/h with modern liquid-handlers and MALDI-TOF systems.

MS-based ligand binding assays with speed, sensitivity, and specificity.

Immunoassays are widely used in biochemical/clinical laboratories owing to their simplicity, speed, and sensitivity. We combined self-assembled monolayer-based immunoassays with MALDI-TOF MS to show that high-fidelity surface preparations with a novel matrix deposition/crystallization technique permits quantitative analysis of monolayer-bound antigens at picomolar detection limits. Calibration curves for intact proteins are possible over a broad concentration range and improved specificity of MS-immunoassays is highlighted by simultaneous label-free quantitation of ligand-bound protein complexes.

Thin-layer matrix sublimation with vapor-sorption induced co-crystallization for sensitive and reproducible SAMDI-TOF MS analysis of protein biosensors.

Coupling immunoassays on self-assembled monolayers (SAMs) to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) provides improved assay selectivity compared with traditional photometric detection techniques. We show that thin-layer-transfer (TLT) of α-cyano-4-hydroxycinnaminic acid (CHCA) MALDI matrix via vacuum sublimation followed by organic solvent-based vapor-sorption induced co-crystallization (VIC) results in unique matrix/analyte co-crystallization tendencies that optimizes assay reproducibility and sensitivity. Unique matrix crystal morphologies resulted from VIC solvent vapors, indicating nucleation and crystal growth characteristics depend upon VIC parameters. We observed that CHCA microcrystals generated by methanol VIC resulted in >10× better sensitivity, increased analyte charging, and improved precision compared with dried droplet measurements. The uniformity of matrix/analyte co-crystallization across planar immunoassays directed at intact proteins yielded low spectral variation for single shot replicates (18.5 % relative standard deviation, RSD) and signal averaged spectra (<10 % RSD). We envision that TLT and VIC for MALDI-TOF will enable high-throughput, reproducible array-based immunoassays for protein molecular diagnostic assays in diverse biochemical and clinical applications.