Orbitrap Mass Spectrometry and High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Enable the in-Depth Analysis of Human Serum Proteoforms.

Blood serum and plasma are arguably the most commonly analyzed clinical samples, with dozens of proteins serving as validated biomarkers for various human diseases. Top-down proteomics may provide additional insights into disease etiopathogenesis since this approach focuses on protein forms, or proteoforms, originally circulating in blood, potentially providing access to information about relevant post-translational modifications, truncations, single amino acid substitutions, and many other sources of protein variation. However, the vast majority of proteomic studies on serum and plasma are carried out using peptide-centric, bottom-up approaches that cannot recapitulate the original proteoform content of samples. Clinical laboratories have been slow to adopt top-down analysis, also due to higher sample handling requirements. In this study, we describe a straightforward protocol for intact proteoform sample preparation based on the depletion of albumin and immunoglobulins, followed by simplified protein fractionation via polyacrylamide gel electrophoresis. After molecular weight-based fractionation, we supplemented the traditional liquid chromatography-tandem mass spectrometry (LC-MS2) data acquisition with high-field asymmetric waveform ion mobility spectrometry (FAIMS) to further simplify serum proteoform mixtures. This LC-FAIMS-MS2 method led to the identification of over 1000 serum proteoforms < 30 kDa, outperforming traditional LC-MS2 data acquisition and more than doubling the number of proteoforms identified in previous studies.

Improved Label-Free Quantification of Intact Proteoforms Using Field Asymmetric Ion Mobility Spectrometry.

The high-throughput quantification of intact proteoforms using a label-free approach is typically performed on proteins in the 0-30 kDa mass range extracted from whole cell or tissue lysates. Unfortunately, even when high-resolution separation of proteoforms is achieved by either high-performance liquid chromatography or capillary electrophoresis, the number of proteoforms that can be identified and quantified is inevitably limited by the inherent sample complexity. Here, we benchmark label-free quantification of proteoforms of Escherichia coli by applying gas-phase fractionation (GPF) via field asymmetric ion mobility spectrometry (FAIMS). Recent advances in Orbitrap instrumentation have enabled the acquisition of high-quality intact and fragmentation mass spectra without the need for averaging time-domain transients prior to Fourier transform. The resulting speed improvements allowed for the application of multiple FAIMS compensation voltages in the same liquid chromatography-tandem mass spectrometry experiment without increasing the overall data acquisition cycle. As a result, the application of FAIMS to label-free quantification based on intact mass spectra substantially increases the number of both identified and quantified proteoforms without penalizing quantification accuracy in comparison to traditional label-free experiments that do not adopt GPF.

Pull-Down of Metalloproteins in Their Native States by Using Desthiobiotin-Based Probes.

One-third of all proteins are estimated to require metals for structural stability and/or catalytic activity. Desthiobiotin probes containing metal binding groups can be used to capture metalloproteins with exposed active-site metals under mild conditions so as to prevent changes in metallation state. The proof-of-concept was demonstrated with carbonic anhydrase (CA), an open active site, Zn2+ -containing protein. CA was targeted by using sulfonamide derivatives. Linkers of various lengths and structures were screened to determine the optimal structure for capture of the native protein. The optimized probes could selectively pull down CA from red blood cell lysate and other protein mixtures. Pull-down of differently metallated CAs was also investigated.

Top-Down Characterization of Heavily Modified Histones Using 193 nm Ultraviolet Photodissociation Mass Spectrometry.

The characterization of protein post-translational modifications (PTMs) remains a significant challenge for traditional bottom-up proteomics methods owing to the lability of PTMs and the difficulty of mapping combinatorial patterns of PTMs based on analysis of small peptides. These shortcomings have accelerated interest in top-down MS/MS methods that focus on analysis of intact proteins. Simultaneous mapping of all PTMs requires extensive sequence coverage to confidently localize modifications. 193 nm ultraviolet photodissociation (UVPD) has been shown to generate unparalleled sequence coverage for intact proteins compared to traditional MS/MS methods. This study focuses on identification and localization of PTMs of histones by UVPD, higher-energy collisional dissociation (HCD), and the hybrid method electron-transfer/higher-energy collision dissociation (EThcD) via a high throughput liquid chromatography-mass spectrometry strategy. In total, over 500 proteoforms were characterized among these three activation methods with 46% of the identifications found in common by two or more activation methods. EThcD and UVPD afforded more extensive characterization of proteoforms than HCD with average gains in sequence coverage of 15% and C-scores that doubled on average.

Extending Proteome Coverage by Combining MS/MS Methods and a Modified Bioinformatics Platform Adapted for Database Searching of Positive and Negative Polarity 193 nm Ultraviolet Photodissociation Mass Spectra.

To extend proteome coverage obtained from bottom-up mass spectrometry approaches, three complementary ion activation methods, higher energy collision dissociation (HCD), ultraviolet photodissociation (UVPD), and negative mode UVPD (NUVPD), are used to interrogate the tryptic peptides in a human hepatocyte lysate using a high performance Orbitrap mass spectrometer. The utility of combining results from multiple activation techniques (HCD+UVPD+NUVPD) is analyzed for total depth and breadth of proteome coverage. This study also benchmarks a new version of the Byonic algorithm, which has been customized for database searches of UVPD and NUVPD data. Searches utilizing the customized algorithm resulted in over 50% more peptide identifications for UVPD and NUVPD tryptic peptide data sets compared to other search algorithms. Inclusion of UVPD and NUVPD spectra resulted in over 600 additional protein identifications relative to HCD alone.

Extensive Characterization of Heavily Modified Histone Tails by 193 nm Ultraviolet Photodissociation Mass Spectrometry via a Middle-Down Strategy.

The ability to map combinatorial patterns of post-translational modifications (PTMs) of proteins remains challenging for traditional bottom-up mass spectrometry workflows. There are also hurdles associated with top-down approaches related to limited data analysis options for heavily modified proteoforms. These shortcomings have accelerated interest in middle-down MS methods that focus on analysis of large peptides generated by specific proteases in conjunction with validated bioinformatics strategies to allow quantification of isomeric histoforms. Mapping multiple PTMs simultaneously requires the ability to obtain high sequence coverage to allow confident localization of the modifications, and 193 nm ultraviolet photodissociation (UVPD) has been shown to cause extensive fragmentation for large peptides and proteins. Histones are an ideal system to test the ability of UVPD to characterize multiple modifications, as the combinations of PTMs are the underpinning of the biological significance of histones and at the same time create an imposing challenge for characterization. The present study focuses on applying 193 nm UVPD to the identification and localization of PTMs on histones by UVPD and comparison to a popular alternative, electron-transfer dissociation (ETD), via a high-throughput middle-down LC/MS/MS strategy. Histone Coder and IsoScale, bioinformatics tools for verification of PTM assignments and quantification of histone peptides, were adapted for UVPD data and applied in the present study. In total, over 300 modified forms were identified, and the distributions of PTMs were quantified between UVPD and ETD. Significant differences in patterns of PTMs were found for histones from HeLa cells prior to and after treatment with a deacetylase inhibitor. Additional fragment ion types generated by UVPD proved essential for extensive characterization of the most heavily modified forms (>5 PTMs).

Pinpointing Double Bond and sn-Positions in Glycerophospholipids via Hybrid 193 nm Ultraviolet Photodissociation (UVPD) Mass Spectrometry.

Complete structural characterization of complex lipids, such as glycerophospholipids, by tandem mass spectrometry (MS/MS) continues to present a major challenge. Conventional activation methods do not generate fragmentation patterns that permit the simultaneous discernment of isomers which differ in both the positions of acyl chains on the glycerol backbone and the double bonds within the acyl chains. Herein we describe a hybrid collisional activation/UVPD workflow that yields near-complete structural information for glycerophospholipids. This hybrid MS3 strategy affords the lipid's sum composition based on the accurate mass measured for the intact lipid as well as highly specific diagnostic product ions that reveal both the acyl chain assignment (i.e., sn-position) and the site-specific location of double bonds in the acyl chains. This approach is demonstrated to differentiate sn-positional and double-bond-positional isomers, such as the regioisomeric phosphatidylcholines PC 16:0/18:1(n-9) and PC 18:1(n-9)/16:0, and has been integrated into an LC-MS3 workflow.

Integrating Carbamylation and Ultraviolet Photodissociation Mass Spectrometry for Middle-Down Proteomics.

The most popular bottom-up proteomics workflow uses trypsin to enzymatically cleave proteins C-terminal to lysine and arginine residues prior to LCMS/MS analysis of the resulting peptides. The high frequency of these residues generates short peptides, some of which are too small or uninformative for optimal analysis and which potentially contribute to gaps in sequence coverage of proteins. Analysis of larger peptides, termed "middle-down", has the potential to span greater sections of protein sequences if the larger peptides are adequately characterized based on their fragmentation patterns. We describe a strategy to generate larger peptides in conjunction with successful characterization by ultraviolet photodissociation (UVPD) for MS/MS analysis in a middle-down workflow, as demonstrated for proteins from E. coli lysates. The larger peptides are produced via modification of lysine residues by carbamylation of proteins. Carbamylation of proteins followed by tryptic digestion produced peptides similar to those expected from Arg-C proteolysis, yet with fewer missed and nonspecific cleavages. UVPD provides excellent sequence coverage of the larger peptides that are often less well characterized by traditional collision-based activation methods.

Impact of Protease on Ultraviolet Photodissociation Mass Spectrometry for Bottom-up Proteomics.

Recent mass spectrometric studies have reported enhanced proteome coverage by employing multiple proteases or by using multiple or alternative activation methods such as electron-transfer dissociation in combination with collisional-activated dissociation (CAD). In this study the use of 193 nm ultraviolet photodissociation for the analysis of thousands of Halobacterium salinarum peptides generated by four proteases (trypsin, LysC, GluC, and chymotrypsin) was evaluated in comparison with higher energy CAD (HCD). Proteins digested by trypsin resulted in greater sequence coverage for HCD over UVPD. LysC digestion resulted in similar sequence coverages for UVPD and HCD; however, for proteins digested by GluC and chymotrypsin 5-10% more sequence coverage on average was achieved by UVPD. HCD resulted in more peptide identifications (at 1% false discovery rate) for trypsin (4356 peptides by HCD versus 3907 peptides by UVPD), whereas UVPD identified greater numbers of peptides for LysC digests (1033 peptides by UVPD versus 844 HCD), chymotrypsin digests (3219 peptides for UVPD versus 2921 for HCD), and GluC digests (2834 peptides for UVPD and 2393 for HCD) and correspondingly greater numbers of proteins.

Improvement of shotgun proteomics in the negative mode by carbamylation of peptides and ultraviolet photodissociation mass spectrometry.

Although acidic peptides compose a substantial portion of many proteomes, their less efficient ionization during positive polarity electrospray ionization (ESI) impedes their detection in bottom-up mass spectrometry workflows. We have implemented a derivatization strategy based on carbamylation which converts basic amine sites (Lys, N-termini) to less basic amides for enhanced analysis in the negative mode. Ultraviolet photodissociation (UVPD) is used to analyze the resulting peptide anions, as demonstrated for tryptic peptides from bovine serum albumin and Halobacterium salinarum in a high throughput liquid chromatography/tandem mass spectrometry (LC/MS/MS) mode. LC/UVPD-MS of a carbamylated H. salinarum digest resulted in 45% more identified peptides and 25% more proteins compared to the unmodified digest analyzed in the negative mode.

A new probe for detecting zinc-bound carbonic anhydrase in cell lysates and cells.

We report the synthesis and application of a small molecule probe for carbonic anhydrase (CA) to track holo-CA in cell lysates and live-cell models of zinc dyshomeostasis. The probe displays a 12-fold increase in fluorescence upon binding to bovine CA and also responds to human CA isoforms.

Modulation of Protein Fragmentation Through Carbamylation of Primary Amines.

We evaluate the impact of carbamylation of the primary amines of the side-chains of Lys and the N-termini on the fragmentation of intact protein ions and the chromatographic properties of a mixture of E. coli ribosomal proteins. The fragmentation patterns of the six unmodified and carbamylated proteins obtained by higher energy collision dissociation (HCD) and ultraviolet photodissociation (UVPD) were compared. Carbamylation significantly reduced the total number of protons retained by the protein owing to the conversion of basic primary amines to non-basic carbamates. Carbamylation caused a significant negative impact on fragmentation of the protein by HCD (i.e., reduced sequence coverage and fewer diagnostic fragment ions) consistent with the mobile proton model, which correlates peptide fragmentation with charge distribution and the opportunity for charge-directed pathways. In addition, fragmentation was enhanced near the N- and C-termini upon HCD of carbamylated proteins. For LCMS/MS analysis of E. coli ribosomal proteins, the retention times increased by 16 min on average upon carbamylation, an outcome attributed to the increased hydrophobicity of the proteins after carbamylation. As noted for both the six model proteins and the ribosomal proteins, carbamylation had relatively little impact on the distribution or types of fragment ions product by UVPD, supporting the proposition that the mechanism of UVPD for intact proteins does not reflect the mobile proton model. Graphical Abstract ᅟ.