Integration of Segmented Ion Fractionation and Differential Ion Mobility on a Q-Exactive Hybrid Quadrupole Orbitrap Mass Spectrometer.

High-field asymmetric waveform ion mobility spectrometry (FAIMS) has gained popularity in the proteomics field for its capability to improve mass spectrometry sensitivity and to decrease peptide co-fragmentation. The recent implementation of FAIMS on Tribrid Orbitrap instruments enhanced proteome coverage and increased the precision of quantitative measurements. However, the FAIMS interface has not been available on older generation Orbitrap mass spectrometers such as the Q-Exactive. Here, we report the integration of the FAIMS Pro device with embedded electrical and gas connections to a Q-Exactive HF mass spectrometer. Proteomic experiments performed on HeLa tryptic digests with the modified mass spectrometer improved signal to noise and reduced interfering ions, resulting in an increase of 42% in peptide identification. FAIMS was also combined with segmented ion fractionation where 100 m/z windows were obtained in turn to further increase the depth of proteome analysis by reducing the proportion of chimeric MS/MS spectra from 50 to 27%. We also demonstrate the application of FAIMS to improve quantitative measurements when using isobaric peptide labeling. FAIMS experiments performed on a two-proteome model revealed that FAIMS Pro provided a 65% improvement in quantification accuracy compared to conventional LC-MS/MS experiments.

Benchmarking the Orbitrap Tribrid Eclipse for Next Generation Multiplexed Proteomics.

The rise of sample multiplexing in quantitative proteomics for the dissection of complex phenotypic comparisons has been advanced by the development of ever more sensitive and robust instrumentation. Here, we evaluated the utility of the Orbitrap Eclipse Tribrid mass spectrometer (advanced quadrupole filter, optimized FTMS scan overhead) and new instrument control software features (Precursor Fit filtering, TurboTMT and Real-time Peptide Search filtering). Multidimensional comparisons of these novel features increased total peptide identifications by 20% for SPS-MS3 methods and 14% for HRMS2 methods. Importantly Real-time Peptide Search filtering enabled a ∼2× throughput improvement for quantification. Across the board, these sensitivity increases were attained without sacrificing quantitative accuracy. New hardware and software features enable more efficient characterization in pursuit of comparative whole proteome insights.

Characterization and Optimization of Multiplexed Quantitative Analyses Using High-Field Asymmetric-Waveform Ion Mobility Mass Spectrometry.

Multiplexed, isobaric tagging methods are powerful techniques to increase throughput, precision, and accuracy in quantitative proteomics. The dynamic range and accuracy of quantitation, however, can be limited by coisolation of tag-containing peptides that release reporter ions and conflate quantitative measurements across precursors. Methods to alleviate these effects often lead to the loss of protein and peptide identifications through online or offline filtering of interference containing spectra. To alleviate this effect, high-Field Asymmetric-waveform Ion Mobility Spectroscopy (FAIMS) has been proposed as a method to reduce precursor coisolation and improve the accuracy and dynamic range of multiplex quantitation. Here we tested the use of FAIMS to improve quantitative accuracy using previously established TMT-based interference standards (triple-knockout [TKO] and Human-Yeast Proteomics Resource [HYPER]). We observed that FAIMS robustly improved the quantitative accuracy of both high-resolution MS2 (HRMS2) and synchronous precursor selection MS3 (SPS-MS3)-based methods without sacrificing protein identifications. We further optimized and characterized the main factors that enable robust use of FAIMS for multiplexed quantitation. We highlight these factors and provide method recommendations to take advantage of FAIMS technology to improve isobaric-tag-quantification moving forward.

A Novel Differential Ion Mobility Device Expands the Depth of Proteome Coverage and the Sensitivity of Multiplex Proteomic Measurements.

The depth of proteomic analyses is often limited by the overwhelming proportion of confounding background ions that compromise the identification and quantification of low abundance peptides. To alleviate these limitations, we present a new high field asymmetric waveform ion mobility spectrometry (FAIMS) interface that can be coupled to the Orbitrap Tribrid mass spectrometers. The interface provides several advantages over previous generations of FAIMS devices, including ease of operation, robustness, and high ion transmission. Replicate LC-FAIMS-MS/MS analyses (n = 100) of HEK293 protein digests showed stable ion current over extended time periods with uniform peptide identification on more than 10,000 distinct peptides. For complex tryptic digest analyses, the coupling of FAIMS to LC-MS/MS enabled a 30% gain in unique peptide identification compared with non-FAIMS experiments. Improvement in sensitivity facilitated the identification of low abundance peptides, and extended the limit of detection by almost an order of magnitude. The reduction in chimeric MS/MS spectra using FAIMS also improved the precision and the number of quantifiable peptides when using isobaric labeling with tandem mass tag (TMT) 10-plex reagent. We compared quantitative proteomic measurements for LC-MS/MS analyses performed using synchronous precursor selection (SPS) and LC-FAIMS-MS/MS to profile the temporal changes in protein abundance of HEK293 cells following heat shock for periods up to 9 h. FAIMS provided 2.5-fold increase in the number of quantifiable peptides compared with non-FAIMS experiments (30,848 peptides from 2,646 proteins for FAIMS versus 12,400 peptides from 1,229 proteins with SPS). Altogether, the enhancement in ion transmission and duty cycle of the new FAIMS interface extended the depth and comprehensiveness of proteomic analyses and improved the precision of quantitative measurements.

Comprehensive Single-Shot Proteomics with FAIMS on a Hybrid Orbitrap Mass Spectrometer.

Liquid chromatography (LC) prefractionation is often implemented to increase proteomic coverage; however, while effective, this approach is laborious, requires considerable sample amount, and can be cumbersome. We describe how interfacing a recently described high-field asymmetric waveform ion mobility spectrometry (FAIMS) device between a nanoelectrospray ionization (nanoESI) emitter and an Orbitrap hybrid mass spectrometer (MS) enables the collection of single-shot proteomic data with comparable depth to that of conventional two-dimensional LC approaches. This next generation FAIMS device incorporates improved ion sampling at the ESI-FAIMS interface, increased electric field strength, and a helium-free ion transport gas. With fast internal compensation voltage (CV) stepping (25 ms/transition), multiple unique gas-phase fractions may be analyzed simultaneously over the course of an MS analysis. We have comprehensively demonstrated how this device performs for bottom-up proteomics experiments as well as characterized the effects of peptide charge state, mass loading, analysis time, and additional variables. We also offer recommendations for the number of CVs and which CVs to use for different lengths of experiments. Internal CV stepping experiments increase protein identifications from a single-shot experiment to >8000, from over 100 000 peptide identifications in as little as 5 h. In single-shot 4 h label-free quantitation (LFQ) experiments of a human cell line, we quantified 7818 proteins with FAIMS using intra-analysis CV switching compared to 6809 without FAIMS. Single-shot FAIMS results also compare favorably with LC fractionation experiments. A 6 h single-shot FAIMS experiment generates 8007 protein identifications, while four fractions analyzed for 1.5 h each produce 7776 protein identifications.

Building ProteomeTools based on a complete synthetic human proteome.

We describe ProteomeTools, a project building molecular and digital tools from the human proteome to facilitate biomedical research. Here we report the generation and multimodal liquid chromatography-tandem mass spectrometry analysis of >330,000 synthetic tryptic peptides representing essentially all canonical human gene products, and we exemplify the utility of these data in several applications. The resource (available at http://www.proteometools.org) will be extended to >1 million peptides, and all data will be shared with the community via ProteomicsDB and ProteomeXchange.

Neucode Labels for Multiplexed, Absolute Protein Quantification.

We describe a new method to accomplish multiplexed, absolute protein quantification in a targeted fashion. The approach draws upon the recently developed neutron encoding (NeuCode) metabolic labeling strategy and parallel reaction monitoring (PRM). Since PRM scanning relies upon high-resolution tandem mass spectra for targeted protein quantification, incorporation of multiple NeuCode labeled peptides permits high levels of multiplexing that can be accessed from high-resolution tandem mass spectra. Here we demonstrate this approach in cultured cells by monitoring a viral infection and the corresponding viral protein production over many infection time points in a single experiment. In this context the NeuCode PRM combination affords up to 30 channels of quantitative information in a single MS experiment.

High-resolution filtering for improved small molecule identification via GC/MS.

Gas chromatography/mass spectrometry (GC/MS) has long been considered one of the premiere analytical tools for small molecule analysis. Recently, a number of GC/MS systems equipped with high-resolution mass analyzers have been introduced. These systems provide analysts with a new dimension of information, accurate mass measurement to the third or fourth decimal place; however, existing data processing tools do not capitalize on this information. Beyond that, GC/MS spectral reference libraries, which have been curated over the last several decades, contain almost exclusively unit resolution MS spectra making integration of accurate mass data dubious. Here we present an informatic approach, called high-resolution filtering (HRF), which bridges this gap. During HRF, high-resolution mass spectra are assigned putative identifications through traditional spectral matching at unit resolution. Once candidate identities have been assigned, all unique combinations of atoms from these candidate precursors are generated and matched to m/z peaks using narrow mass tolerances. The total amount of measured signal that is annotated is used as a metric of plausibility for the presumed identification. Here we demonstrate that the HRF approach is both feasible and highly specific toward correct identifications.

The Negative Mode Proteome with Activated Ion Negative Electron Transfer Dissociation (AI-NETD).

The field of proteomics almost uniformly relies on peptide cation analysis, leading to an underrepresentation of acidic portions of proteomes, including relevant acidic posttranslational modifications. Despite the many benefits negative mode proteomics can offer, peptide anion analysis remains in its infancy due mainly to challenges with high-pH reversed-phase separations and a lack of robust fragmentation methods suitable for peptide anion characterization. Here, we report the first implementation of activated ion negative electron transfer dissociation (AI-NETD) on the chromatographic timescale, generating 7,601 unique peptide identifications from Saccharomyces cerevisiae in single-shot nLC-MS/MS analyses of tryptic peptides-a greater than 5-fold increase over previous results with NETD alone. These improvements translate to identification of 1,106 proteins, making this work the first negative mode study to identify more than 1,000 proteins in any system. We then compare the performance of AI-NETD for analysis of peptides generated by five proteases (trypsin, LysC, GluC, chymotrypsin, and AspN) for negative mode analyses, identifying as many as 5,356 peptides (1,045 proteins) with LysC and 4,213 peptides (857 proteins) with GluC in yeast-characterizing 1,359 proteins in total. Finally, we present the first deep-sequencing approach for negative mode proteomics, leveraging offline low-pH reversed-phase fractionation prior to online high-pH separations and peptide fragmentation with AI-NETD. With this platform, we identified 3,467 proteins in yeast with trypsin alone and characterized a total of 3,730 proteins using multiple proteases, or nearly 83% of the expressed yeast proteome. This work represents the most extensive negative mode proteomics study to date, establishing AI-NETD as a robust tool for large-scale peptide anion characterization and making the negative mode approach a more viable platform for future proteomic studies.

One-hour proteome analysis in yeast.

Recent advances in chromatography and mass spectrometry (MS) have made rapid and deep proteomic profiling possible. To maximize the performance of the recently produced Orbitrap hybrid mass spectrometer, we have developed a protocol that combines improved sample preparation (including optimized cellular lysis by extensive bead beating) and chromatographic conditions (specifically, 30-cm capillary columns packed with 1.7-µm bridged ethylene hybrid material) and the manufacture of a column heater (to accommodate flow rates of 350-375 nl/min) that increases the number of proteins identified across a single liquid chromatography-tandem MS (LC-MS/MS) separation, thereby reducing the need for extensive sample fractionation. This strategy allowed the identification of up to 4,002 proteins (at a 1% false discovery rate (FDR)) in yeast (Saccharomyces cerevisiae strain BY4741) over 70 min of LC-MS/MS analysis. Quintuplicate analysis of technical replicates reveals 83% overlap at the protein level, thus demonstrating the reproducibility of this procedure. This protocol, which includes cell lysis, overnight tryptic digestion, sample analysis and database searching, takes ∼24 h to complete. Aspects of this protocol, including chromatographic separation and instrument parameters, can be adapted for the optimal analysis of other organisms.

NeuCode labels for relative protein quantification.

We describe a synthesis strategy for the preparation of lysine isotopologues that differ in mass by as little as 6 mDa. We demonstrate that incorporation of these molecules into the proteomes of actively growing cells does not affect cellular proliferation, and we discuss how to use the embedded mass signatures (neutron encoding (NeuCode)) for multiplexed proteome quantification by means of high-resolution mass spectrometry. NeuCode SILAC amalgamates the quantitative accuracy of SILAC with the multiplexing of isobaric tags and, in doing so, offers up new opportunities for biological investigation. We applied NeuCode SILAC to examine the relationship between transcript and protein levels in yeast cells responding to environmental stress. Finally, we monitored the time-resolved responses of five signaling mutants in a single 18-plex experiment.

Organic acid quantitation by NeuCode methylamidation.

We have developed a multiplexed quantitative analysis method for carboxylic acids by liquid chromatography high resolution mass spectrometry. The method employs neutron encoded (NeuCode) methylamine labels ((13)C or (15)N enriched) that are affixed to carboxylic acid functional groups to enable duplex quantitation via mass defect measurement. This work presents the first application of NeuCode quantitation to small molecules. We have applied this technique to detect adulteration of olive oil by quantitative analysis of fatty acid methyl amide derivatives, and the quantitative accuracy of the NeuCode analysis was validated by GC/MS. Currently, the method enables duplex quantitation and is expandable to at least 6-plex analysis.

Intelligent data acquisition blends targeted and discovery methods.

A mass spectrometry (MS) method is described here that can reproducibly identify hundreds of peptides across multiple experiments. The method uses intelligent data acquisition to precisely target peptides while simultaneously identifying thousands of other, nontargeted peptides in a single nano-LC-MS/MS experiment. We introduce an online peptide elution order alignment algorithm that targets peptides based on their relative elution order, eliminating the need for retention-time-based scheduling. We have applied this method to target 500 mouse peptides across six technical replicate nano-LC-MS/MS experiments and were able to identify 440 of these in all six, compared with only 256 peptides using data-dependent acquisition (DDA). A total of 3757 other peptides were also identified within the same experiment, illustrating that this hybrid method does not eliminate the novel discovery advantages of DDA. The method was also tested on a set of mice in biological quadruplicate and increased the number of identified target peptides in all four mice by over 80% (826 vs 459) compared with the standard DDA method. We envision real-time data analysis as a powerful tool to improve the quality and reproducibility of proteomic data sets.

Neutron-encoded mass signatures for quantitative top-down proteomics.

The ability to acquire highly accurate quantitative data is an increasingly important part of any proteomics experiment, whether shotgun or top-down approaches are used. We recently developed a quantitation strategy for peptides based on neutron encoding, or NeuCode SILAC, which uses closely spaced heavy isotope-labeled amino acids and high-resolution mass spectrometry to provide quantitative data. We reasoned that the strategy would also be applicable to intact proteins and could enable robust, multiplexed quantitation for top-down experiments. We used yeast lysate labeled with either (13)C6(15)N2-lysine or (2)H8-lysine, isotopologues of lysine that are spaced 36 mDa apart. Proteins having such close spacing cannot be distinguished during a medium resolution scan, but upon acquiring a high-resolution scan, the two forms of the protein with each amino acid are resolved and the quantitative information revealed. An additional benefit NeuCode SILAC provides for top down is that the spacing of the isotope peaks indicates the number of lysines present in the protein, information that aids in identification. We used NeuCode SILAC to quantify several hundred isotope distributions, manually identify and quantify proteins from 1:1, 3:1, and 5:1 mixed ratios, and demonstrate MS(2)-based quantitation using ETD.

The one hour yeast proteome.

We describe the comprehensive analysis of the yeast proteome in just over one hour of optimized analysis. We achieve this expedited proteome characterization with improved sample preparation, chromatographic separations, and by using a new Orbitrap hybrid mass spectrometer equipped with a mass filter, a collision cell, a high-field Orbitrap analyzer, and, finally, a dual cell linear ion trap analyzer (Q-OT-qIT, Orbitrap Fusion). This system offers high MS(2) acquisition speed of 20 Hz and detects up to 19 peptide sequences within a single second of operation. Over a 1.3 h chromatographic method, the Q-OT-qIT hybrid collected an average of 13,447 MS(1) and 80,460 MS(2) scans (per run) to produce 43,400 (x) peptide spectral matches and 34,255 (x) peptides with unique amino acid sequences (1% false discovery rate (FDR)). On average, each one hour analysis achieved detection of 3,977 proteins (1% FDR). We conclude that further improvements in mass spectrometer scan rate could render comprehensive analysis of the human proteome within a few hours.

Amine-reactive neutron-encoded labels for highly plexed proteomic quantitation.

We describe a novel amine-reactive chemical label that exploits differential neutron-binding energy between (13)C and (15)N isotopes. These neutron-encoded (NeuCode) chemical labels enable up to 12-plex MS1-based protein quantification. Each structurally identical, but isotopically unique, tag is encoded with a 12.6-mDa mass difference-relative to its nearest neighbor-so that peptides bearing these NeuCode signatures do not increase spectral complexity and are detected only upon analysis with very high mass-resolving powers. We demonstrate that the method provides quantitative performance that is comparable to both metabolic labeling and isobaric tagging while combining the benefits of both strategies. Finally, we employ the tags to characterize the proteome of Saccharomyces cerevisiae during the diauxic shift, a metabolic transition from fermentation to aerobic respiration.

Neutron encoded labeling for peptide identification.

Metabolic labeling of cells using heavy amino acids is most commonly used for relative quantitation; however, partner mass shifts also detail the number of heavy amino acids contained within the precursor species. Here, we use a recently developed metabolic labeling technique, NeuCode (neutron encoding) stable isotope labeling with amino acids in cell culture (SILAC), which produces precursor partners spaced ~40 mDa apart to enable amino acid counting. We implement large scale counting of amino acids through a program, "Amino Acid Counter", which determines the most likely combination of amino acids within a precursor based on NeuCode SILAC partner spacing and filters candidate peptide sequences during a database search using this information. Counting the number of lysine residues for precursors selected for MS/MS decreases the median number of candidate sequences from 44 to 14 as compared to an accurate mass search alone (20 ppm). Furthermore, the ability to co-isolate and fragment NeuCode SILAC partners enables counting of lysines in product ions, and when the information is used, the median number of candidates is reduced to 7. We then demonstrate counting leucine in addition to lysine results in a 6-fold decrease in search space, 43 to 7, when compared to an accurate mass search. We use this scheme to analyze a nanoLC-MS/MS experiment and demonstrate that accurate mass plus lysine and leucine counting reduces the number of candidate sequences to one for ~20% of all precursors selected, demonstrating an ability to identify precursors without MS/MS analysis.

Neutron-encoded mass signatures for multiplexed proteome quantification.

We describe a protein quantification method called neutron encoding that exploits the subtle mass differences caused by nuclear binding energy variation in stable isotopes. These mass differences are synthetically encoded into amino acids and incorporated into yeast and mouse proteins via metabolic labeling. Mass spectrometry analysis with high mass resolution (>200,000) reveals the isotopologue-embedded peptide signals, permitting quantification. Neutron encoding will enable highly multiplexed proteome analysis with excellent dynamic range and accuracy.

Calorie restriction and SIRT3 trigger global reprogramming of the mitochondrial protein acetylome.

Calorie restriction (CR) extends life span in diverse species. Mitochondria play a key role in CR adaptation; however, the molecular details remain elusive. We developed and applied a quantitative mass spectrometry method to probe the liver mitochondrial acetyl-proteome during CR versus control diet in mice that were wild-type or lacked the protein deacetylase SIRT3. Quantification of 3,285 acetylation sites-2,193 from mitochondrial proteins-rendered a comprehensive atlas of the acetyl-proteome and enabled global site-specific, relative acetyl occupancy measurements between all four experimental conditions. Bioinformatic and biochemical analyses provided additional support for the effects of specific acetylation on mitochondrial protein function. Our results (1) reveal widespread reprogramming of mitochondrial protein acetylation in response to CR and SIRT3, (2) identify three biochemically distinct classes of acetylation sites, and (3) provide evidence that SIRT3 is a prominent regulator in CR adaptation by coordinately deacetylating proteins involved in diverse pathways of metabolism and mitochondrial maintenance.