Spatial top-down proteomics for the functional characterization of human kidney.

Background: The Human Proteome Project has credibly detected nearly 93% of the roughly 20,000 proteins which are predicted by the human genome. However, the proteome is enigmatic, where alterations in amino acid sequences from polymorphisms and alternative splicing, errors in translation, and post-translational modifications result in a proteome depth estimated at several million unique proteoforms. Recently mass spectrometry has been demonstrated in several landmark efforts mapping the human proteoform landscape in bulk analyses. Herein, we developed an integrated workflow for characterizing proteoforms from human tissue in a spatially resolved manner by coupling laser capture microdissection, nanoliter-scale sample preparation, and mass spectrometry imaging. Results: Using healthy human kidney sections as the case study, we focused our analyses on the major functional tissue units including glomeruli, tubules, and medullary rays. After laser capture microdissection, these isolated functional tissue units were processed with microPOTS (microdroplet processing in one-pot for trace samples) for sensitive top-down proteomics measurement. This provided a quantitative database of 616 proteoforms that was further leveraged as a library for mass spectrometry imaging with near-cellular spatial resolution over the entire section. Notably, several mitochondrial proteoforms were found to be differentially abundant between glomeruli and convoluted tubules, and further spatial contextualization was provided by mass spectrometry imaging confirming unique differences identified by microPOTS, and further expanding the field-of-view for unique distributions such as enhanced abundance of a truncated form (1-74) of ubiquitin within cortical regions. Conclusions: We developed an integrated workflow to directly identify proteoforms and reveal their spatial distributions. Where of the 20 differentially abundant proteoforms identified as discriminate between tubules and glomeruli by microPOTS, the vast majority of tubular proteoforms were of mitochondrial origin (8 of 10) where discriminate proteoforms in glomeruli were primarily hemoglobin subunits (9 of 10). These trends were also identified within ion images demonstrating spatially resolved characterization of proteoforms that has the potential to reshape discovery-based proteomics because the proteoforms are the ultimate effector of cellular functions. Applications of this technology have the potential to unravel etiology and pathophysiology of disease states, informing on biologically active proteoforms, which remodel the proteomic landscape in chronic and acute disorders.

Transient isotachophoresis-Capillary zone electrophoresis-Mass spectrometry method with off-line microscale solid phase extraction pretreatment for quantitation of intact low molecular mass proteins in various biological fluids.

Quantification of proteins is still predominantly done by the traditional bottom-up approach. Targeting of intact proteins in complex biological matrices is connected with multiple challenges during the sample pretreatment, separation, and detection step of the analytical workflow. In this work, we focused on the development of an on-line hyphenated capillary zone electrophoresis-mass spectrometry method employing off-line microscale solid-phase extraction based on hydrophilic lipophilic balance (HLB) sorbent as a sample pretreatment step for the analysis of low molecular mass intact proteins (<20 kDa) spiked in various biological fluids (human serum, plasma, urine, and saliva). A detailed optimization process involved the selection of a suitable capillary surface, background electrolyte (BGE), and comparison of two in-capillary preconcentration methods, namely transient isotachophoresis (tITP) and dynamic pH junction (DPJ), to enhance the sensitivity of the method. Optimum separation of the analytes was achieved using uncoated bare fused silica capillary employing 500 mM formic acid (pH 1.96) + 5 % (v/v) acetonitrile as BGE. tITP was utilized as an optimum preconcentration technique, achieving a 19- to 127-fold increase in the signal intensity when using 200 mM ammonium formate (adjusted to pH 4.00) as the leading electrolyte and BGE as the terminating electrolyte. Off-line microscale solid-phase extraction with various eluate treatment procedures was evaluated to ensure the compatibility of the sample pretreatment method with the selected in-capillary preconcentration, separation, and detection process. Achieved extraction recoveries of spiked proteins were in the range of 76-100 % for urine, 12-54 % for serum, 21-106 % for plasma, and 25-98 % for saliva when the eluate was evaporated and reconstituted in the solution of the leading electrolyte to achieve the tITP process. The optimum method was validated across different biological matrices, offering good linearity, accuracy, and precision, and making it suitable for proteomic studies (e.g., therapeutic drug monitoring, biomarker research) in different biological samples.

Liquid Extraction Surface Analysis Mass Spectrometry Imaging of Denatured Intact Proteins.

Liquid extraction surface analysis (LESA) is an ambient surface sampling technique that can be coupled with mass spectrometry (MS) to analyze analytes directly from biological substrates such as tissue sections. LESA MS involves liquid microjunction sampling of a substrate by use of a discrete volume of solvent followed by nano-electrospray ionization. As the technique makes use of electrospray ionization, it lends itself to the analysis of intact proteins. Here, we describe the use of LESA MS to analyze and image the distribution of intact denatured proteins from thin fresh frozen tissue sections.

Combining offline high performance liquid chromatography fractionation of peptides and intact proteins to enhance proteome coverage in bottom-up proteomics.

Offline peptide separation (PS) using high-performance liquid chromatography (HPLC) is currently used to enhance liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection of proteins. In search of more effective methods for enhancing MS proteome coverage, we developed a robust method for intact protein separation (IPS), an alternative first-dimension separation technique, and explored additional benefits that it offers. Comparing IPS to the traditional PS method, we found that both enhance detection of unique protein IDs to a similar magnitude, though in diverse ways. IPS was especially effective in serum, which has a small number of extremely high abundance proteins. PS was more effective in tissues with fewer dominating high-abundance proteins and was more effective in enhancing detection of post-translational modifications (PTMs). Combining the IPS and PS methods together (IPS+PS) was especially beneficial, enhancing proteome detection more than either method could independently. The comparison of IPS+PS versus six PS fractionation pools increased total number of proteins IDs by nearly double, while also significantly increasing number of unique peptides detected per protein, percent peptide sequence coverage of each protein, and detection of PTMs. This IPS+PS combined method requires fewer LC-MS/MS runs than current PS methods would need to obtain similar improvements in proteome detection, and it is robust, time- and cost-effective, and generally applicable to various tissue and sample types.

Influence of ion-pairing reagents on the separation of intact glycoproteins using hydrophilic-interaction liquid chromatography - high-resolution mass spectrometry.

Hydrophilic-interaction liquid chromatography (HILIC) of intact proteins offers high-resolution separations of glycoforms of glycoproteins differing in the number of (neutral) glycans. However, to obtain efficient separations it is essential that the positively charged sites of the proteins are shielded by acidic (negative) ion-pair reagents (IPRs), so as to enhance the contribution of the hydroxyl groups of the (neutral) sugars in the glycoprotein. Here, we studied the influence of various IPRs that differ in physico-chemical properties, such as hydrophobicity and acidity, on the capillary-scale HILIC separation of intact (glyco)proteins. We evaluated the use of fluoroacetic acid (MFA), difluoroacetic acid (DFA), trifluoroacetic acid (TFA), and heptafluorobutyric acid (HFBA) as diluents for sample preparation, as solvents for sample loading on a reversed-phase trap prior to the HILIC separation, and as mobile-phase components for HILIC and HILIC-MS. To reduce the contribution of ion-exchange interaction with the (silica-based) stationary phase, we used an acrylamide-based monolithic column. We studied the influence of the different IPRs on each step of the separation of a mixture of proteins of different size and hydrophilicity and on the separation of the five glycoforms of ribonuclease B. The content of IPR in the sample was shown not to affect the separation and the MS detection. However, a low content of TFA and DFA in the mobile phase is favourable, as it reduces adduct formation and leads to higher signal intensity. The optimized HILIC conditions successfully resolved nine major glycoforms groups of a ∼40 kDa glycoprotein horseradish peroxidase (HRP), as an example of a complex glycoprotein.

Analysis of intact proteins with capillary zone electrophoresis coupled to mass spectromery using uncoated and coated capillaries.

Although, in general, the application of coated capillaries is recommended for the separation of intact proteins, bare silica capillary is still the most often used capillary due to its simplicity and cheapness. In this work, the performance of bare fused silica capillary for intact protein analysis was compared to that of different (dynamically coated polybrene (PB) and permanently coated linear polyacrylamide (LPA)) coated capillaries using capillary zone electrophoresis - mass spectrometry (CZE-MS). In cases where low pH (pH=1.8) was used in bare silica capillaries, good precision (0.56-0.78 RSD% and 1.7-6.5 RSD% for migration times and peak areas, respectively), minimal adsorption and separation efficiency (N= 27 000/m - 322 000/m) similar to or even better than those obtained with the coated capillaries (created by an intricate multi-step process) was achieved. The PB and the LPA capillaries demonstrated their slightly better resolving power in terms of separating the different forms/variants of the same protein (e.g., hemoglobin subunits). Among the studied capillaries the one with LPA coating showed the most stable separations in the long term (n=25: 0.18-0.49 RSD% and 3.1-4.9 RSD% for migration times and peak areas, respectively). For the separation of a few proteins or even a larger number of proteins in biological samples (e.g., snake venom) the application of the simple and cheap bare fused silica capillary can be considered as an efficient choice.

Evaluating the Leucine Trigger Hypothesis to Explain the Post-prandial Regulation of Muscle Protein Synthesis in Young and Older Adults: A Systematic Review.

Background: The "leucine trigger" hypothesis was originally conceived to explain the post-prandial regulation of muscle protein synthesis (MPS). This hypothesis implicates the magnitude (amplitude and rate) of post-prandial increase in blood leucine concentrations for regulation of the magnitude of MPS response to an ingested protein source. Recent evidence from experimental studies has challenged this theory, with reports of a disconnect between blood leucine concentration profiles and post-prandial rates of MPS in response to protein ingestion. Aim: The primary aim of this systematic review was to qualitatively evaluate the leucine trigger hypothesis to explain the post-prandial regulation of MPS in response to ingested protein at rest and post-exercise in young and older adults. We hypothesized that experimental support for the leucine trigger hypothesis will depend on age, exercise status (rest vs. post-exercise), and type of ingested protein (i.e., isolated proteins vs. protein-rich whole food sources). Methods: This qualitative systematic review extracted data from studies that combined measurements of post-prandial blood leucine concentrations and rates of MPS following ingested protein at rest and following exercise in young and older adults. Data relating to blood leucine concentration profiles and post-prandial MPS rates were extracted from all studies, and reported as providing sufficient or insufficient evidence for the leucine trigger hypothesis. Results: Overall, 16 of the 29 eligible studies provided sufficient evidence to support the leucine trigger hypothesis for explaining divergent post-prandial rates of MPS in response to different ingested protein sources. Of these 16 studies, 13 were conducted in older adults (eight of which conducted measurements post-exercise) and 14 studies included the administration of isolated proteins. Conclusion: This systematic review underscores the merits of the leucine trigger hypothesis for the explanation of the regulation of MPS. However, our data indicate that the leucine trigger hypothesis confers most application in regulating the post-prandial response of MPS to ingested proteins in older adults. Consistent with our hypothesis, we provide data to support the idea that the leucine trigger hypothesis is more relevant within the context of ingesting isolated protein sources rather than protein-rich whole foods. Future mechanistic studies are warranted to understand the complex series of modulatory factors beyond blood leucine concentration profiles within a food matrix that regulate post-prandial rates of MPS.

Top-Down Proteomics of Medicinal Cannabis.

The revised legislation on medicinal cannabis has triggered a surge of research studies in this space. Yet, cannabis proteomics is lagging. In a previous study, we optimised the protein extraction of mature buds for bottom-up proteomics. In this follow-up study, we developed a top-down mass spectrometry (MS) proteomics strategy to identify intact denatured protein from cannabis apical buds. After testing different source-induced dissociation (SID), collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and electron transfer dissociation (ETD) parameters on infused known protein standards, we devised three LC-MS/MS methods for top-down sequencing of cannabis proteins. Different MS/MS modes produced distinct spectra, albeit greatly overlapping between SID, CID, and HCD. The number of fragments increased with the energy applied; however, this did not necessarily translate into greater sequence coverage. Some precursors were more amenable to fragmentation than others. Sequence coverage decreased as the mass of the protein increased. Combining all MS/MS data maximised amino acid (AA) sequence coverage, achieving 73% for myoglobin. In this experiment, most cannabis proteins were smaller than 30 kD. A total of 46 cannabis proteins were identified with 136 proteoforms bearing different post-translational modifications (PTMs), including the excision of N-terminal M, the N-terminal acetylation, methylation, and acetylation of K resides, and phosphorylation. Most identified proteins are involved in photosynthesis, translation, and ATP production. Only one protein belongs to the phytocannabinoid biosynthesis, olivetolic acid cyclase.

Qualitative evaluation of high pH mass spectrometry-compatible reversed phase liquid chromatography for altered selectivity in separations of intact proteins.

Intact proteins are increasingly being recognized as potential biomarkers and biotherapeutic agents for cancer and other serious diseases. Low pH reversed phase plays an important role in both single and multidimensional protein separations for resolving complex protein samples prior to mass spectrometric detection. In this work, we evaluated the use of high pH reversed phase liquid chromatography as an alternative chromatographic separation to gain different selectivity while maintaining the high resolving power and MS compatibility of reversed phase separations. The altered selectivity gained by high pH reversed phase liquid chromatography can further help to separate unresolved protein peaks or to increase peak capacity and resolving power of a multidimensional setup for complex biological samples. Hence, we evaluated the use of different MS-friendly buffers, ion pairing reagents, and stationary phases (silica- and polymer-based) at alkaline pH for intact protein separations. The best chromatographic separation, with complementary selectivity to low pH reversed phase, was achieved using triethylammonium bicarbonate at pH 10 and hybrid silica particles.

In situ synthesis of a monolithic material with multi-sized pores and its chromatographic properties for the separation of intact proteins from human plasma.

The fabrication of a monolithic allyl phenoxyacetate-based material was proposed via the in situ radical polymerization using ethylene dimethacrylate as the crosslinker and 2,2'-azobisisobutyronitrile as the initiator within a stainless steel column (50 mm × 4.6 mm i.d.). The effects of the porogen composition, the crosslinker amount and the monomer type on the resulting monoliths were investigated. The morphology of the monoliths was characterized using scanning electron microscopy and a nitrogen adsorption-desorption instrument, and the pore structure was characterized using mercury intrusion porosimetry. The results indicate that the optimized monolith has a micro-, meso- and macro- multi-sized pore structure with a high specific surface area of 260.66 m2 g-1. The resulting monoliths were used as stationary phases for the separation of proteins from bio-samples, including a mixture of six standard proteins, chicken egg whites, snailase and human plasma, using high-performance liquid chromatography. Compared to optimized glycidyl methacrylate-based and styrene-based monolithic columns, the allyl phenoxyacetate-based monolithic column exhibited improved selectivity in the separation of proteins. Furthermore, the present method avoids the masking of highly abundant proteins, such as human serum albumin, immunoglobulin G and human fibrinogen, in the detection of middle- or low-abundance proteins in human plasma. The protein identification results obtained from liquid chromatography/mass spectrometry indicate that the present method is an outstanding method for efficient fractionation of human plasma, which will be especially useful in future plasma proteomics research.

Fabrication of a monolithic, macroporous diallyl maleate-based material and its application for fast separation of intact proteins from human plasma with reversed-phase chromatography.

A monolithic diallyl maleate-based column was developed and used to separate intact proteins from complex bio-samples with high-performance liquid chromatography. The resulting monolith exhibited a relatively uniform porous structure with macro-pores. A sample of standard proteins mixture was used to explore the reversed-phase mechanism for fast separation on the homemade monolithic column. The observed retention time increased with the values of the diameters, relative hydrophobicities and molecular weights of the standard proteins, which mainly depends on the relative hydrophobicity of the proteins. It is worth mentioning that the mask of the three most abundant proteins in human plasma can be avoided, thus providing an opportunity for the middle- and low-abundance proteins to be detected. According to this exploration, the fast separation of human plasma proteins on the diallyl maleate-based monolithic column was achieved in 15 min. The chromatographic fractions were identified by liquid chromatography-tandem mass spectrometry, and the results indicate that the present method is an outstanding method for the fast and efficient fractionation of human plasma that will be significant sense for plasma proteomics research, especially for exploring new disease marker and drug target.

Fast, selective and quantitative protein profiling of adenovirus-vector based vaccines by ultra-performance liquid chromatography.

A method for the quantitative determination of the protein composition of adenovirus-vector based vaccines was developed. The final method used RP-UPLC with UV absorbance detection, a C4 column (300 Å, 1.7 µm, 2.1 × 150 mm), and a water- acetonitrile (ACN) gradient containing trifluoroacetic acid (TFA) as ion-pairing agent. The chromatographic resolution between the various adenovirus proteins was optimized by studying the effect of the TFA concentration and the column temperature, applying a full factorial design of experiments. A reproducible baseline separation of all relevant adenovirus proteins could be achieved within 17 min run time. Samples containing adenovirus particles could be directly injected into the UPLC system without sample pretreatment. The viruses reproducibly dissociate into proteins in the UPLC system upon contact with the mobile phase containing ACN. The new RP-UPLC method was successfully validated for protein profiling and relative quantification of proteins in vaccine products based on adenovirus vector types 26 and 35. The intermediate precision of the relative peak areas of all proteins was between 1% and 14% RSD, except for the peak assigned to protein V (26% RSD). The method proved to be stability indicating with respect to thermal and oxidation stress of the adenovirus-vector based vaccine and was successfully implemented for the characterization of adenovirus-based products.

Ambient ionisation mass spectrometry for in situ analysis of intact proteins.

Ambient surface mass spectrometry is an emerging field which shows great promise for the analysis of biomolecules directly from their biological substrate. In this article, we describe ambient ionisation mass spectrometry techniques for the in situ analysis of intact proteins. As a broad approach, the analysis of intact proteins offers unique advantages for the determination of primary sequence variations and posttranslational modifications, as well as interrogation of tertiary and quaternary structure and protein-protein/ligand interactions. In situ analysis of intact proteins offers the potential to couple these advantages with information relating to their biological environment, for example, their spatial distributions within healthy and diseased tissues. Here, we describe the techniques most commonly applied to in situ protein analysis (liquid extraction surface analysis, continuous flow liquid microjunction surface sampling, nano desorption electrospray ionisation, and desorption electrospray ionisation), their advantages, and limitations and describe their applications to date. We also discuss the incorporation of ion mobility spectrometry techniques (high field asymmetric waveform ion mobility spectrometry and travelling wave ion mobility spectrometry) into ambient workflows. Finally, future directions for the field are discussed.

Accelerated SDS depletion from proteins by transmembrane electrophoresis: Impacts of Joule heating.

SDS plays a key role in proteomics workflows, including protein extraction, solubilization and mass-based separations (e.g. SDS-PAGE, GELFrEE). However, SDS interferes with mass spectrometry and so it must be removed prior to analysis. We recently introduced an electrophoretic platform, termed transmembrane electrophoresis (TME), enabling extensive depletion of SDS from proteins in solution with exceptional protein yields. However, our prior TME runs required 1 h to complete, being limited by Joule heating which causes protein aggregation at higher operating currents. Here, we demonstrate effective strategies to maintain lower TME sample temperatures, permitting accelerated SDS depletion. Among these strategies, the use of a magnetic stir bar to continuously agitate a model protein system (BSA) allows SDS to be depleted below 100 ppm (>98% removal) within 10 min of TME operations, while maintaining exceptional protein recovery (>95%). Moreover, these modifications allow TME to operate without any user intervention, improving throughput and robustness of the approach. Through fits of our time-course SDS depletion curves to an exponential model, we calculate SDS depletion half-lives as low as 1.2 min. This promising electrophoretic platform should provide proteomics researchers with an effective purification strategy to enable MS characterization of SDS-containing proteins.

Sequencing Larger Intact Proteins (30-70 kDa) with Activated Ion Electron Transfer Dissociation.

The analysis of intact proteins via mass spectrometry can offer several benefits to proteome characterization, although the majority of top-down experiments focus on proteoforms in a relatively low mass range (<30 kDa). Recent studies have focused on improving the analysis of larger intact proteins (up to ~75 kDa), but they have also highlighted several challenges to be addressed. One major hurdle is the efficient dissociation of larger protein ions, which often to do not yield extensive fragmentation via conventional tandem MS methods. Here we describe the first application of activated ion electron transfer dissociation (AI-ETD) to proteins in the 30-70 kDa range. AI-ETD leverages infrared photo-activation concurrent to ETD reactions to improve sequence-informative product ion generation. This method generates more product ions and greater sequence coverage than conventional ETD, higher-energy collisional dissociation (HCD), and ETD combined with supplemental HCD activation (EThcD). Importantly, AI-ETD provides the most thorough protein characterization for every precursor ion charge state investigated in this study, making it suitable as a universal fragmentation method in top-down experiments. Additionally, we highlight several acquisition strategies that can benefit characterization of larger proteins with AI-ETD, including combination of spectra from multiple ETD reaction times for a given precursor ion, multiple spectral acquisitions of the same precursor ion, and combination of spectra from two different dissociation methods (e.g., AI-ETD and HCD). In all, AI-ETD shows great promise as a method for dissociating larger intact protein ions as top-down proteomics continues to advance into larger mass ranges. Graphical Abstract ᅟ.

Highly efficient enrichment of low-abundance intact proteins by core-shell structured Fe3O4-chitosan@graphene composites.

In proteomics research, the screening and monitoring of disease biomarkers is still a major challenge, mainly due to their low concentration in biological samples. However, the universal enrichment of intact proteins has not been further studied. In this work, we developed a Fe3O4-chitosan@graphene (Fe3O4-CS@G) core-shell composite to enrich low-abundance proteins from biological samples. Fe3O4-CS@G composite holds chitosan layer decorated Fe3O4 core, which improves the hydrophilicity of materials greatly. Meanwhile, the graphene nanosheets shell formed via electrostatic assembly endows the composite with huge surface area (178m2/g). The good water dispersibility ensures the sufficient contact opportunities between graphene composites and proteins, and the large surface area provides enough adsorption sites for the enrichment of proteins. Using Fe3O4-CS@G, four standard proteins Cyt-c, BSA, Myo and OVA were enriched with better adsorption capacity and recovery rate, compared with previously reported magnetic graphene composites. Additionally, the mechanism of compared to" is corrected into "compared with". proteins adsorption on Fe3O4-CS@G was further studied, which indicates that hydrophobic and electrostatic interaction work together to facilitate the universal and efficient enrichment of proteins. Human plasma sample was employed to further evaluate the enrichment performance of Fe3O4-CS@G. Eventually, 123 proteins were identified from one of SAX fractions of human plasma, which is much better than commercial Sep-pak C18 enrichment column (39 proteins). All these outstanding performances suggest that Fe3O4-CS@G is an ideal platform for the enrichment of low-abundance intact proteins and thus holds great potential to facilitate the identification of biomarkers from biological samples in proteomics research.

Activated Ion-Electron Transfer Dissociation Enables Comprehensive Top-Down Protein Fragmentation.

Here we report the first demonstration of near-complete sequence coverage of intact proteins using activated ion-electron transfer dissociation (AI-ETD), a method that leverages concurrent infrared photoactivation to enhance electron-driven dissociation. AI-ETD produces mainly c/z-type product ions and provides comprehensive (77-97%) protein sequence coverage, outperforming HCD, ETD, and EThcD for all proteins investigated. AI-ETD also maintains this performance across precursor ion charge states, mitigating charge-state dependence that limits traditional approaches.

Different Stationary Phase Selectivities and Morphologies for Intact Protein Separations.

The central dogma of biology proposed that one gene encodes for one protein. We now know that this does not reflect reality. The human body has approximately 20,000 protein-encoding genes; each of these genes can encode more than one protein. Proteins expressed from a single gene can vary in terms of their post-translational modifications, which often regulate their function within the body. Understanding the proteins within our bodies is a key step in understanding the cause, and perhaps the solution, to disease. This is one of the application areas of proteomics, which is defined as the study of all proteins expressed within an organism at a given point in time. The human proteome is incredibly complex. The complexity of biological samples requires a combination of technologies to achieve high resolution and high sensitivity analysis. Despite the significant advances in mass spectrometry, separation techniques are still essential in this field. Liquid chromatography is an indispensable tool by which low-abundant proteins in complex samples can be enriched and separated. However, advances in chromatography are not as readily adapted in proteomics compared to advances in mass spectrometry. Biologists in this field still favour reversed-phase chromatography with fully porous particles. The purpose of this review is to highlight alternative selectivities and stationary phase morphologies that show potential for application in top-down proteomics; the study of intact proteins.

High-resolution ultrahigh-pressure long column reversed-phase liquid chromatography for top-down proteomics.

Separation of proteoforms for global intact protein analysis (i.e. top-down proteomics) has lagged well behind what is achievable for peptides in traditional bottom-up proteomic approach and is becoming a true bottle neck for top-down proteomics. Herein, we report use of long (≥1M) columns containing short alkyl (C1-C4) bonded phases to achieve high-resolution RPLC for separation of proteoforms. At a specific operation pressure limit (i.e., 96.5MPa or 14Kpsi used in this work), column length was found to be the most important factor for achieving maximal resolution separation of proteins when 1.5-5µm particles were used as packings and long columns provided peak capacities greater than 400 for proteoforms derived from a global cell lysate with molecular weights below 50kDa. Larger proteoforms (50-110kDa) were chromatographed on long RPLC columns and detected by MS; however, they cannot be identified yet by tandem mass spectrometry. Our experimental data further demonstrated that long alkyl (e.g., C8 and C18) bonded particles provided high-resolution RPLC for <10kDa proteoforms, not efficient for separation of global proteoforms. Reversed-phase particles with porous, nonporous, and superficially porous surfaces were systematically investigated for high-resolution RPLC. Pore size (200-400Å) and the surface structure (porous and superficially porous) of particles was found to have minor influences on high-resolution RPLC of proteoforms. RPLC presented herein enabled confident identification of ∼900 proteoforms (1% FDR) for a low-microgram quantity of proteomic samples using a single RPLC-MS/MS analysis. The level of RPLC performance attained in this work is close to that typically realized in bottom-up proteomics, and broadly useful when applying e.g., the single-stage MS accurate mass tag approach, but less effective when combined with current tandem MS. Our initial data indicate that MS detection and fragmentation inefficiencies provided by current high-resolution mass spectrometers are key challenges for characterization of larger proteoforms.

Reversed-phase separation parameters for intact proteins using liquid chromatography with triple quadrupole mass spectrometry.

The separation of intact proteins is inherently more complex than that of small molecules using reversed-phase liquid chromatography. The goal of this work was to determine a reasonable set of operational parameters (a recommended starting point for other analysts) for the separation of intact proteins and their detection by triple quadrupole mass spectrometry. Although protein separations have been studied for many years, the direct detection of intact proteins with mass spectrometry requires special considerations of mobile phase additives to achieve efficient separation and sensitive detection. Myoglobin, cytochrome c, lactalbumin, lysozyme, and ubiquitin were used as model analytes to investigate chromatographic method development using a triple quadrupole mass spectrometer and detection by multiple reaction monitoring. Chromatographic parameters including the concentration of trifluoroacetic acid, flow rate, gradient slope, temperature, mobile phase composition, and stationary phase chemistry were evaluated. Protein charge state profiles were also monitored for temperature and modifier effects. An optimized method using 0.2 mL/min flow rate, 15% gradient slope, and 75°C with a combined trifluoroacetic acid and formic acid modified mobile phase was developed.