Direct Tissue Profiling of Protein Complexes: Toward Native Mass Spectrometry Imaging

Native mass spectrometry seeks to probe noncovalent protein interactions in terms of protein quaternary structure, protein–protein and protein–ligand complexes. The ultimate goal is to link the understanding of protein interactions to the protein environment by visualizing the spatial distribution of noncovalent protein interactions within tissue. Previously, we have shown that noncovalently bound protein complexes can be directly probed via liquid extraction surface analysis from dried blood spot samples, where hemoglobin is highly abundant. Here, we show that the intact hemoglobin complex can be sampled directly from thin tissue sections of mouse liver and correlated to a visible vascular feature, paving the way for native mass spectrometry imaging

High Field Asymmetric Waveform Ion Mobility Spectrometry in Nontargeted Bottom-up Proteomics of Dried Blood Spots

Despite the great potential of dried blood spots (DBS) as a source of endogenous proteins for biomarker discovery, the literature relating to nontargeted bottom-up proteomics of DBS is sparse, primarily due to the inherent complexity and very high dynamic range associated with these samples. Here, we present proof-of-concept results in which we have coupled high field asymmetric waveform ion mobility spectrometry (FAIMS) with liquid chromatography–tandem mass spectrometry (LC–MS/MS) for nontargeted bottom-up proteomics of DBS with the aim of addressing these challenges. We, and others, have previously demonstrated the benefits of FAIMS more generally in proteomics including improved signal-to-noise and extended proteome coverage, and the aim of the current work was to extend those benefits specifically to DBS. The DBS samples were either extracted by the more traditional manual “punch and elute” approach or by an automated liquid surface extraction (LESA) approach prior to trypsin digestion. The resulting samples were analyzed by LC–MS/MS and LC–FAIMS–MS/MS analysis. The results show that the total number of proteins identified increased by ∼50% for the punch and elute samples and ∼45% for the LESA samples in the LC–FAIMS–MS/MS analysis. For both the punch and elute samples and the LESA samples, ∼30% of the total proteins identified were observed in both the LC–MS/MS and the LC–FAIMS–MS/MS data sets, illustrating the complementarity of the approaches. Overall, this work demonstrates the benefits of inclusion of FAIMS for nontargeted proteomics of DBS

High Field Asymmetric Waveform Ion Mobility Spectrometry in Nontargeted Bottom-up Proteomics of Dried Blood Spots

Despite the great potential of dried blood spots (DBS) as a source of endogenous proteins for biomarker discovery, the literature relating to nontargeted bottom-up proteomics of DBS is sparse, primarily due to the inherent complexity and very high dynamic range associated with these samples. Here, we present proof-of-concept results in which we have coupled high field asymmetric waveform ion mobility spectrometry (FAIMS) with liquid chromatography–tandem mass spectrometry (LC–MS/MS) for nontargeted bottom-up proteomics of DBS with the aim of addressing these challenges. We, and others, have previously demonstrated the benefits of FAIMS more generally in proteomics including improved signal-to-noise and extended proteome coverage, and the aim of the current work was to extend those benefits specifically to DBS. The DBS samples were either extracted by the more traditional manual “punch and elute” approach or by an automated liquid surface extraction (LESA) approach prior to trypsin digestion. The resulting samples were analyzed by LC–MS/MS and LC–FAIMS–MS/MS analysis. The results show that the total number of proteins identified increased by ∼50% for the punch and elute samples and ∼45% for the LESA samples in the LC–FAIMS–MS/MS analysis. For both the punch and elute samples and the LESA samples, ∼30% of the total proteins identified were observed in both the LC–MS/MS and the LC–FAIMS–MS/MS data sets, illustrating the complementarity of the approaches. Overall, this work demonstrates the benefits of inclusion of FAIMS for nontargeted proteomics of DBS

Subcritical Water Processing of Proteins: An Alternative to Enzymatic Digestion?

Subcritical water is an emerging tool in the processing of bioorganic waste. Subcritical water is an environmentally benign solvent which has the potential to provide an alternative to traditional methods of protein hydrolysis without the inclusion of expensive acids or enzymes. To date, most studies on the subcritical water mediated hydrolysis of proteins have focused on the production of amino acids, rather than the intermediate peptides. Here, we investigate the specificity of subcritical water with respect to the production of peptides from three model proteins, hemoglobin, bovine serum albumin, and β-casein, and compare the results with enzymatic digestion of proteins by trypsin. In addition, the effect of subcritical water (SCW) treatment on two protein post-translational modifications, disulfide bonds and phosphorylation, was investigated. The results show that high protein sequence coverages (>80%) can be obtained following subcritical water hydrolysis. These are comparable to those obtained following treatment with tryspin. Under mild subcritical water conditions (160 °C), all proteins showed favored cleavage of the Asp-X bond. The results for β-casein revealed favored cleavage of the Glu-X bond at subcritical water temperatures of 160 and 207 °C. That was similarly observed for bovine serum albumin at a subcritical water temperature of 207 °C. Subcritical water treatment results in very limited cleavage of disulfide bonds. Reduction and alkylation of proteins either prior to or post subcritical water treatment improve reported protein sequence coverages. The results for phosphoprotein β-casein show that, under mild subcritical water conditions, phosphorylation may be retained on the peptide hydrolysis products

Large-Scale Analysis of Peptide Sequence Variants: The Case for High-Field Asymmetric Waveform Ion Mobility Spectrometry

Large scale analysis of proteins by mass spectrometry is becoming increasingly routine; however, the presence of peptide isomers remains a significant challenge for both identification and quantitation in proteomics. Classes of isomers include sequence inversions, structural isomers, and localization variants. In many cases, liquid chromatography is inadequate for separation of peptide isomers. The resulting tandem mass spectra are composite, containing fragments from multiple precursor ions. The benefits of high-field asymmetric waveform ion mobility spectrometry (FAIMS) for proteomics have been demonstrated by a number of groups, but previously work has focused on extending proteome coverage generally. Here, we present a systematic study of the benefits of FAIMS for a key challenge in proteomics, that of peptide isomers. We have applied FAIMS to the analysis of a phosphopeptide library comprising the sequences GPSGXVpSXAQLX­(K/R) and SXPFKXpSPLXFG­(K/R), where X = ADEFGLSTVY. The library has defined limits enabling us to make valid conclusions regarding FAIMS performance. The library contains numerous sequence inversions and structural isomers. In addition, there are large numbers of theoretical localization variants, allowing false localization rates to be determined. The FAIMS approach is compared with reversed-phase liquid chromatography and strong cation exchange chromatography. The FAIMS approach identified 35% of the peptide library, whereas LC–MS/MS alone identified 8% and LC–MS/MS with strong cation exchange chromatography prefractionation identified 17.3% of the library

Large-Scale Analysis of Peptide Sequence Variants: The Case for High-Field Asymmetric Waveform Ion Mobility Spectrometry

Large scale analysis of proteins by mass spectrometry is becoming increasingly routine; however, the presence of peptide isomers remains a significant challenge for both identification and quantitation in proteomics. Classes of isomers include sequence inversions, structural isomers, and localization variants. In many cases, liquid chromatography is inadequate for separation of peptide isomers. The resulting tandem mass spectra are composite, containing fragments from multiple precursor ions. The benefits of high-field asymmetric waveform ion mobility spectrometry (FAIMS) for proteomics have been demonstrated by a number of groups, but previously work has focused on extending proteome coverage generally. Here, we present a systematic study of the benefits of FAIMS for a key challenge in proteomics, that of peptide isomers. We have applied FAIMS to the analysis of a phosphopeptide library comprising the sequences GPSGXVpSXAQLX­(K/R) and SXPFKXpSPLXFG­(K/R), where X = ADEFGLSTVY. The library has defined limits enabling us to make valid conclusions regarding FAIMS performance. The library contains numerous sequence inversions and structural isomers. In addition, there are large numbers of theoretical localization variants, allowing false localization rates to be determined. The FAIMS approach is compared with reversed-phase liquid chromatography and strong cation exchange chromatography. The FAIMS approach identified 35% of the peptide library, whereas LC–MS/MS alone identified 8% and LC–MS/MS with strong cation exchange chromatography prefractionation identified 17.3% of the library

High-Throughput Deconvolution of Native Protein Mass Spectrometry Imaging Data Sets for Mass Domain Analysis

Protein mass spectrometry imaging (MSI) with electrospray-based ambient ionization techniques, such as nanospray desorption electrospray ionization (nano-DESI), generates data sets in which each pixel corresponds to a mass spectrum populated by peaks corresponding to multiply charged protein ions. Importantly, the signal associated with each protein is split among multiple charge states. These peaks can be transformed into the mass domain by spectral deconvolution. When proteins are imaged under native/non-denaturing conditions to retain non-covalent interactions, deconvolution is particularly valuable in helping interpret the data. To improve the acquisition speed, signal-to-noise ratio, and sensitivity, native MSI is usually performed using mass resolving powers that do not provide isotopic resolution, and conventional algorithms for deconvolution of lower-resolution data are not suitable for these large data sets. UniDec was originally developed to enable rapid deconvolution of complex protein mass spectra. Here, we developed an updated feature set harnessing the high-throughput module, MetaUniDec, to deconvolve each pixel of native MSI data sets and transform m/z-domain image files to the mass domain. New tools enable the reading, processing, and output of open format .imzML files for downstream analysis. Transformation of data into the mass domain also provides greater accessibility, with mass information readily interpretable by users of established protein biology tools such as sodium dodecyl sulfate polyacrylamide gel electrophoresis

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High Field Asymmetric Waveform Ion Mobility Spectrometry in Nontargeted Bottom-up Proteomics of Dried Blood Spots

Despite the great potential of dried blood spots (DBS) as a source of endogenous proteins for biomarker discovery, the literature relating to nontargeted bottom-up proteomics of DBS is sparse, primarily due to the inherent complexity and very high dynamic range associated with these samples. Here, we present proof-of-concept results in which we have coupled high field asymmetric waveform ion mobility spectrometry (FAIMS) with liquid chromatography–tandem mass spectrometry (LC–MS/MS) for nontargeted bottom-up proteomics of DBS with the aim of addressing these challenges. We, and others, have previously demonstrated the benefits of FAIMS more generally in proteomics including improved signal-to-noise and extended proteome coverage, and the aim of the current work was to extend those benefits specifically to DBS. The DBS samples were either extracted by the more traditional manual “punch and elute” approach or by an automated liquid surface extraction (LESA) approach prior to trypsin digestion. The resulting samples were analyzed by LC–MS/MS and LC–FAIMS–MS/MS analysis. The results show that the total number of proteins identified increased by ∼50% for the punch and elute samples and ∼45% for the LESA samples in the LC–FAIMS–MS/MS analysis. For both the punch and elute samples and the LESA samples, ∼30% of the total proteins identified were observed in both the LC–MS/MS and the LC–FAIMS–MS/MS data sets, illustrating the complementarity of the approaches. Overall, this work demonstrates the benefits of inclusion of FAIMS for nontargeted proteomics of DBS