Chemical Cross-Linking and Mass Spectrometry As a Low-Resolution Protein Structure Determination Technique

Protein complexes are the foundation of a majority of cellular processes. Although a large number of protein complexes have been identified through biochemical experiments, the precise molecular details and three-dimensional structures are available for only a small fraction. Chemical cross-linking coupled with mass spectrometry (CXMS) has gained popularity in recent years for characterization of inter- and intraprotein interactions in protein complexes. This perspective provides a comprehensive and critical overview of CXMS strategies employed for structural elucidation of protein complexes. We evaluate the challenges associated with CXMS techniques with special emphasis on data analysis. As sensitivity, mass resolution, mass accuracy and ease of use of mass spectrometers have improved, the complexity of processing and interpreting CXMS data has become the central problem to be addressed. We review here a number of computer programs available to address these problems

xComb: A Cross-Linked Peptide Database Approach to Protein−Protein Interaction Analysis

We developed an informatic method to identify tandem mass spectra composed of chemically cross-linked peptides from those of linear peptides and to assign sequence to each of the two unique peptide sequences. For a given set of proteins the key software tool, xComb, combs through all theoretically feasible cross-linked peptides to create a database consisting of a subset of all combinations represented as peptide FASTA files. The xComb library of select theoretical cross-linked peptides may then be used as a database that is examined by a standard proteomic search engine to match tandem mass spectral data sets to identify cross-linked peptides. The database search may be conducted against as many as 50 proteins with a number of common proteomic search engines, e.g. Phenyx, Sequest, OMSSA, Mascot and X!Tandem. By searching against a peptide library of linearized, cross-linked peptides, rather than a linearized protein library, search times are decreased and the process is decoupled from any specific search engine. A further benefit of decoupling from the search engine is that protein cross-linking studies may be conducted with readily available informatics tools for which scoring routines already exist within the proteomic community

Mapping PARP‑1 Auto-ADP-ribosylation Sites by Liquid Chromatography–Tandem Mass Spectrometry

We demonstrate a novel method for the identification of poly­(ADP-ribose) polymerase-1 (PARP-1) autopoly­(ADP-ribosyl)­ation sites that is suited to collision induced dissociation (CID) tandem mass spectrometry. By employing phosphodiesterase to remove the majority of the poly­(ADP-ribose) (pADPr) modification, we reduce the complexity of tandem mass spectrometric analysis of pADPr-modified tryptic peptides. The simplified ribose-5′-phosphate form of the peptides produce tandem mass spectra by CID that are readily interpreted and enable effective localization of the exact sites of PARP-1-catalyzed poly­(ADP-ribosyl)­ation. In conjunction with a phosphopeptide-like enrichment strategy that captures the ribose-5′-phosphate peptides, we identified eight novel sites of PARP-1 automodification, confirmed the localization of two sites previously reported, and provided evidence for two additional targeted peptides with ambiguous modification site assignments. Given the simplicity of the approach, the method is readily applicable to analysis of complex samples

On the Relevance of Peptide Sequence Permutations in Shotgun Proteomics Studies

In collision-induced dissociation (CID) of peptides, it has been observed that rearrangement processes can take place that appear to permute/scramble the original primary structure, which may in principle adversely affect peptide identification. Here, an analysis of sequence permutation in tandem mass spectra is presented for a previously published proteomics study on P. aeruginosa (Scherl et al., J. Am. Soc. Mass Spectrom. 2008, 19, 891) conducted using an LTQ-orbitrap. Overall, 4878 precursor ions are matched by considering the accurate mass (i.e., <5 ppm) of the precursor ion and at least one fragment ion that confirms the sequence. The peptides are then grouped into higher- and lower-confidence data sets, using five fragment ions as a cutoff for higher-confidence identification. It is shown that the propensity for sequence permutation increases with the length of the tryptic peptide in both data sets. A higher charge state (i.e., 3+ vs 2+) also appears to correlate with a higher appearance of permuted masses for larger peptides. The ratio of these permuted sequence ions, compared to all tandem mass spectral peaks, reaches ∼25% in the higher-confidence data set, compared to an estimated incidence of false positives for permuted masses (maximum ∼8%), based on a null-hypothesis decoy data set

Surface Acoustic Wave Nebulization of Peptides As a Microfluidic Interface for Mass Spectrometry

We describe the fabrication of a surface acoustic wave (SAW) device on a LiNbO3 piezoelectric transducer for the transfer of nonvolatile analytes to the gas phase at atmospheric pressure (a process referred to as nebulization or atomization). We subsequently show how such a device can be used in the field of mass spectrometry (MS) detection, demonstrating that SAW nebulization (SAWN) can be performed either in a discontinuous or pulsed mode, similar to that for matrix assisted laser desorption ionization (MALDI) or in a continuous mode like electrospray ionization (ESI). We present data showing the transfer of peptides to the gas phase, where ions are detected by MS. These peptide ions were subsequently fragmented by collision-induced dissociation, from which the sequence was assigned. Unlike MALDI mass spectra, which are typically contaminated with matrix ions at low m/z, the SAWN generated spectra had no such interference. In continuous mode, the SAWN plume was sampled on a microsecond time scale by a linear ion trap mass spectrometer and produced multiply charged peptide precursor ions with a charge state distribution shifted to higher m/z compared to an identical sample analyzed by ESI. The SAWN technology also provides the opportunity to re-examine a sample from a flat surface, repeatedly. The process can be performed without the need for capillaries, which can clog, reservoirs, which dilute the sample, and electrodes, which when in direct contact with sample, cause unwanted electrochemical oxidation. In both continuous and pulsed sampling modes, the quality of precursor ion scans and tandem mass spectra of peptides was consistent across the plume’s lifetime

Faster, Quantitative, and Accurate Precursor Acquisition Independent From Ion Count

Data-dependent precursor ion selection is widely used in shotgun proteomics to profile the protein components of complex samples. Although very popular, this bottom-up method presents major drawbacks in terms of detectable dynamic range. Recently, we demonstrated the superior performance of a data-independent method we termed precursor acquisition independent from ion count (PAcIFIC). Here, we report a faster, accurate, multiplexed, and quantitative PAcIFIC method. Our results show that the time needed to perform such analysis can be decreased by 33% to 66% using modern ion trap instruments and that high mass accuracy can be applied to such a strategy. Quantification capability is demonstrated on protein standards and a whole bacterial cell lysate using isobaric tagging. Finally, we confirm in yeast the dynamic range capabilities of such a method where proteins down to less than 50 copies per cell can be monitored without sample prefractionation

A Sensitive GC-MS Method for Quantitation of Lipid A Backbone Components and Terminal Phosphate Modifications

Lipid A, the hydrophobic anchor of lipopolysaccharide (LPS) present in the outer membrane of Gram-negative bacteria, serves as a target for cationic antimicrobial peptides, such as polymyxins. Membrane stress from polymyxins results in activation of two-component regulatory systems that produce lipid A modifying enzymes. These enzymes add neutral moieties, such as aminoarabinose (AraN) and ethanolamine (EtN) to lipid A terminal phosphates that mask the phosphate’s negative charge and inhibit electrostatic interaction with the cationic polymyxins. Currently, these modifications may be detected by MALDI-TOF MS; however, this analysis is only semiquantitative. Herein we describe a GC-MS method to quantitate lipid A backbone components, glucosamine (GlcN) and inorganic phosphate (Pi), along with terminal phosphate modifications AraN and EtN. In this assay, lipid A is isolated from Gram-negative bacterial samples, hydrolyzed into its individual moieties, and derivatized via methoximation followed by silylation prior to analysis via GC-MS. Changes in AraN and EtN quantity were characterized using a variety of regulatory mutants of Salmonella, revealing differences that were not detected using MALDI-TOF MS analysis. Additionally, an increase in the abundance of AraN and EtN modifications were observed when resistant Enterobacter and Escherichia coli strains were grown in the presence of colistin (polymyxin E). Lastly, increased levels of Pi were found in bisphosphorylated lipid A compared to monophosphorylated lipid A samples. Because lipid A modifications serve as indicators of polymyxin resistance in Gram-negative bacteria, this method provides the capacity to monitor polymyxin resistance by quantification of lipid A modification using GC-MS

Model-Based Spectral Library Approach for Bacterial Identification via Membrane Glycolipids

By circumventing the need for a pure colony, MALDI-TOF mass spectrometry of bacterial membrane glycolipids (lipid A) has the potential to identify microbes more rapidly than protein-based methods. However, currently available bioinformatics algorithms (e.g., dot products) do not work well with glycolipid mass spectra such as those produced by lipid A, the membrane anchor of lipopolysaccharide. To address this issue, we propose a spectral library approach coupled with a machine learning technique to more accurately identify microbes. Here, we demonstrate the performance of the model-based spectral library approach for microbial identification using approximately a thousand mass spectra collected from multi-drug-resistant bacteria. At false discovery rates < 1%, our approach identified many more bacterial species than the existing approaches such as the Bruker Biotyper and characterized over 97% of their phenotypes accurately. As the diversity in our glycolipid mass spectral library increases, we anticipate that it will provide valuable information to more rapidly treat infected patients

Structural Elucidation of Intact Rough-type Lipopolysaccharides Using Field Asymmetric Ion Mobility Spectrometry and Kendrick Mass Defect Plots

Lipopolysaccharides (LPSs) are a hallmark virulence factor of Gram-negative bacteria. They are complex, structurally heterogeneous mixtures due to variations in number, type, and position of their simplest units: fatty acids and monosaccharides. Thus, LPS structural characterization by traditional mass spectrometry (MS) methods is challenging. Here, we describe the benefits of field asymmetric ion mobility spectrometry (FAIMS) for analysis of an intact R-type lipopolysaccharide complex mixture (lipooligosaccharide; LOS). Structural characterization was performed using Escherichia coli J5 (Rc mutant) LOS, a TLR4 agonist widely used in glycoconjugate vaccine research. FAIMS gas-phase fractionation improved the (S/N) ratio and number of detected LOS species. Additionally, FAIMS allowed the separation of overlapping isobars facilitating their tandem MS characterization and unequivocal structural assignments. In addition to FAIMS gas-phase fractionation benefits, extra sorting of the structurally related LOS molecules was further accomplished using Kendrick mass defect (KMD) plots. Notably, a custom KMD base unit of [Na–H] created a highly organized KMD plot that allowed identification of interesting and novel structural differences across the different LOS ion families, i.e., ions with different acylation degrees, oligosaccharides composition, and chemical modifications. Defining the composition of a single LOS ion by tandem MS along with the organized KMD plot structural network was sufficient to deduce the composition of 181 LOS species out of 321 species present in the mixture. The combination of FAIMS and KMD plots allowed in-depth characterization of the complex LOS mixture and uncovered a wealth of novel information about its structural variations

Structural Elucidation of Intact Rough-type Lipopolysaccharides Using Field Asymmetric Ion Mobility Spectrometry and Kendrick Mass Defect Plots

Lipopolysaccharides (LPSs) are a hallmark virulence factor of Gram-negative bacteria. They are complex, structurally heterogeneous mixtures due to variations in number, type, and position of their simplest units: fatty acids and monosaccharides. Thus, LPS structural characterization by traditional mass spectrometry (MS) methods is challenging. Here, we describe the benefits of field asymmetric ion mobility spectrometry (FAIMS) for analysis of an intact R-type lipopolysaccharide complex mixture (lipooligosaccharide; LOS). Structural characterization was performed using Escherichia coli J5 (Rc mutant) LOS, a TLR4 agonist widely used in glycoconjugate vaccine research. FAIMS gas-phase fractionation improved the (S/N) ratio and number of detected LOS species. Additionally, FAIMS allowed the separation of overlapping isobars facilitating their tandem MS characterization and unequivocal structural assignments. In addition to FAIMS gas-phase fractionation benefits, extra sorting of the structurally related LOS molecules was further accomplished using Kendrick mass defect (KMD) plots. Notably, a custom KMD base unit of [Na–H] created a highly organized KMD plot that allowed identification of interesting and novel structural differences across the different LOS ion families, i.e., ions with different acylation degrees, oligosaccharides composition, and chemical modifications. Defining the composition of a single LOS ion by tandem MS along with the organized KMD plot structural network was sufficient to deduce the composition of 181 LOS species out of 321 species present in the mixture. The combination of FAIMS and KMD plots allowed in-depth characterization of the complex LOS mixture and uncovered a wealth of novel information about its structural variations