Multiply Charged Peptides Research Articles

Electrospray ionization in-source decay of N-glycans and the effects on N-glycan structural identification.

Electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) are soft ionization techniques commonly used in mass spectrometry. Although in-source and post-source decays of MALDI have been investigated extensively, the analogous decays of ESI have received little attention. Previous studies regarding the analogous decays of ESI focus on the dissociation of multiply charged proteins and peptides. The decay of carbohydrates in ESI has not been investigated yet, and it may have interference in carbohydrate structural determination. Commercial apparatus, including a high-performance liquid chromatography (HPLC), an ESI source, and a linear ion trap mass spectrometer, were used to investigate the fragmentation of several N-glycans during the ESI process. About 0.2%-3% of neutral N-glycans and more than 50% of N-glycans consisting of a sialic acid are dissociated into small N-glycans by ESI in-source decay in typical ESI operating conditions. The efficiencies of most dissociation channels increase as the temperature of ion transfer capillary increases, indicating that part of the energy deposited into the precursor ions for cracking is from the heated capillary. The cracking patterns of ESI in-source decay are slightly different from those of gaseous phase collision-induced dissociation. Large N-glycans are dissociated into small N-glycans in ESI in-source decay that may result in the interference of the structural identification of small N-glycans. Separation of large N-glycans from small N-glycans, for example, using HPLC, prior to ESI ionization is necessary to eliminate the interference. This is particularly important when N-glycans consist of sialic acid or large N-glycans have much higher concentration than that of small N-glycans in ESI solution.

Ultrahigh-Throughput Sample Analysis Using Liquid Atmospheric Pressure Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.

Mass spectrometry (MS) allows for automated analysis of complex samples at high resolution without the need for labeling/derivatization. Liquid atmospheric pressure matrix-assisted laser desorption/ionization (LAP-MALDI) enables rapid sample preparation and MS analysis using microtiter-plate formats and high-performing mass spectrometers. We present a step change in high-speed, large-scale MS sample analysis of peptides at 20 samples/s and an enzymatic assay at 40 samples/s, i.e., an order of magnitude faster than current MS platforms. LAP-MALDI requires only low amounts of sample volume (<2 μL), of which only a fraction (<1%) is typically consumed, and allows for multiplexing and high-speed MS/MS analysis, demonstrated at ∼10 samples/s. Its high ion signal stability and similarity to electrospray ionization enables CVs below 10% and the analysis of multiply charged peptide ions at these extreme speeds. LAP-MALDI MS fulfills the speed requirements for large-scale population diagnostics and compound screening with the potential of analyzing >1 million samples per day.

Ultrasensitive single-cell proteomics workflow identifies >1000 protein groups per mammalian cell.

We report on the combination of nanodroplet sample preparation, ultra-low-flow nanoLC, high-field asymmetric ion mobility spectrometry (FAIMS), and the latest-generation Orbitrap Eclipse Tribrid mass spectrometer for greatly improved single-cell proteome profiling. FAIMS effectively filtered out singly charged ions for more effective MS analysis of multiply charged peptides, resulting in an average of 1056 protein groups identified from single HeLa cells without MS1-level feature matching. This is 2.3 times more identifications than without FAIMS and a far greater level of proteome coverage for single mammalian cells than has been previously reported for a label-free study. Differential analysis of single microdissected motor neurons and interneurons from human spinal tissue indicated a similar level of proteome coverage, and the two subpopulations of cells were readily differentiated based on single-cell label-free quantification.

Gas-Phase Enrichment of Multiply Charged Peptide Ions by Differential Ion Mobility Extend the Comprehensiveness of SUMO Proteome Analyses.

The small ubiquitin-like modifier (SUMO) is a member of the family of ubiquitin-like modifiers (UBLs) and is involved in important cellular processes, including DNA damage response, meiosis and cellular trafficking. The large-scale identification of SUMO peptides in a site-specific manner is challenging not only because of the low abundance and dynamic nature of this modification, but also due to the branched structure of the corresponding peptides that further complicate their identification using conventional search engines. Here, we exploited the unusual structure of SUMO peptides to facilitate their separation by high-field asymmetric waveform ion mobility spectrometry (FAIMS) and increase the coverage of SUMO proteome analysis. Upon trypsin digestion, branched peptides contain a SUMO remnant side chain and predominantly form triply protonated ions that facilitate their gas-phase separation using FAIMS. We evaluated the mobility characteristics of synthetic SUMO peptides and further demonstrated the application of FAIMS to profile the changes in protein SUMOylation of HEK293 cells following heat shock, a condition known to affect this modification. FAIMS typically provided a 10-fold improvement of detection limit of SUMO peptides, and enabled a 36% increase in SUMO proteome coverage compared to the same LC-MS/MS analyses performed without FAIMS. Graphical Abstract ᅟ.

Protein identification using a nanoUHPLC-AP-MALDI MS/MS workflow with CID of multiply charged proteolytic peptides

Liquid AP-MALDI can produce predominantly multiply charged ESI-like ions and stable durable analyte ion yields with samples allowing good shot-to-shot reproducibility and exhibiting self-healing properties during laser irradiation. In this study, LC-MALDI MS/MS workflows that utilize multiply charged ions are reported for the first time and compared with standard LC-ESI MS/MS for bottom-up proteomic analysis. The proposed method is compatible with trifluoroacetic acid as an LC ion pairing reagent and allows multiple MS/MS acquisitions of the LC-separated samples without substantial sample consumption. In addition, the method facilitates the storage of fully spotted MALDI target plates for months without significant sample degradation.

Generation of multiply charged peptides and proteins from glycerol-based matrices using lasers with ultraviolet, visible and near-infrared wavelengths and an atmospheric pressure ion source

Conventional matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is characterized by the predominant generation of singly charged analyte ions. Recent studies demonstrated that high charge states of peptides and small proteins can be produced with an atmospheric pressure (AP) MALDI ion source if liquid glycerol-based matrix systems are utilized and the AP-vacuum transfer is realized via a heated ion inlet tube [R. Cramer, A. Pirkl, F. Hillenkamp, K. Dreisewerd, Angew. Chem. Int. Ed. 52 (2013) 2364–2367]. Here, we used this AP ion source and employed an optical parametric oscillator (OPO) laser to study the wavelength dependence of the ion generation between 260 and 1080 nm. Three matrix systems with different optical absorption characteristics were investigated: non-absorbing glycerol mixed with trifluoroacetic acid (TFA) and glycerol mixed with either 2,4- or 2,5-dihydroxybenzoic acids as two classical UV-MALDI matrices. The highest ion yields of multiply charged peptides were consistently obtained at laser wavelengths for which the chromophore-containing matrix systems exhibited the lowest optical absorption. Best sensitivities were even achieved with the transparent glycerol/TFA mixture and by use of a Nd:YAG-laser operated at 1064nm (or with the OPO laser tuned to a similar wavelength). Experiments with absorbing and transparent sample substrates demonstrated the involvement of substrate absorption. In contrast to the results obtained with the AP ion source, using the same matrix systems in combination with a standard oMALDI2™ ion source, operated at a fine vacuum of ∼1mbar and without an inlet tube, the inverse wavelength dependence was observed. Or in other words, like in standard UV-MALDI under these conditions the highest analyte ion yields were obtained for laser excitation wavelengths corresponding to a high absorptivity of the matrix. Our findings thus point to completely different desorption/ionization processes with the two ion source geometries and laser excitation regimes. We hypothesize that for the AP case the material ablation comprises low-energy laser spallation and that rapid evaporative ionization of small droplets in the heated transfer capillary is responsible for charge production. From an analytical point of view, the production of multiply-charged peptide and protein ions with charge states that for the tested analytes (ranging by mass from 1 to 17kDa) almost fully resembled those generated by nano-electrospray ionization (ESI) could facilitate the coupling of laser-based ion sources with mass analyzers, such as orthogonal-extracting time-of-flight (QTOF) instruments or orbitraps which exhibit only limited m/z ranges.

Investigation and optimization of parameters affecting the multiply charged ion yield in AP-MALDI MS

Liquid matrix-assisted laser desorption/ionization (MALDI) allows the generation of predominantly multiply charged ions in atmospheric pressure (AP) MALDI ion sources for mass spectrometry (MS) analysis. The charge state distribution of the generated ions and the efficiency of the ion source in generating such ions crucially depend on the desolvation regime of the MALDI plume after desorption in the AP-to-vacuum inlet. Both high temperature and a flow regime with increased residence time of the desorbed plume in the desolvation region promote the generation of multiply charged ions. Without such measures the application of an electric ion extraction field significantly increases the ion signal intensity of singly charged species while the detection of multiply charged species is less dependent on the extraction field. In general, optimization of high temperature application facilitates the predominant formation and detection of multiply charged compared to singly charged ion species. In this study an experimental set-up and optimization strategy is described for liquid AP-MALDI MS which improves the ionization efficiency of selected ion species up to 14 times. In combination with ion mobility separation, the method allows the detection of multiply charged peptide and protein ions for analyte solution concentrations as low as 2fmol/μL (0.5μL, i.e. 1fmol, deposited on the target) with very low sample consumption in the low nL-range.

Gas-Phase Amidation of Carboxylic Acids with Woodward's Reagent K Ions.

Gas-phase amidation of carboxylic acids in multiply-charged peptides is demonstrated via ion/ion reactions with Woodward's reagent K (wrk) in both positive and negative mode. Woodward's reagent K, N-ethyl-3-phenylisoxazolium-3'-sulfonate, is a commonly used reagent that activates carboxylates to form amide bonds with amines in solution. Here, we demonstrate that the analogous gas-phase chemistry occurs upon reaction of the wrk ions and doubly protonated (or doubly deprotonated) peptide ions containing the carboxylic acid functionality. The reaction involves the formation of the enol ester intermediate in the electrostatic complex. Upon collisional activation, the ethyl amine on the reagent is transferred to the activated carbonyl carbon on the peptide, resulting in the formation of an ethyl amide (addition of 27 Da to the peptide) with loss of a neutral ketene derivative. Further collision-induced dissociation (CID) of the products and comparison with solution-phase amidation product confirms the structure of the ethyl amide.

Selective removal of alkali metal cations from multiply-charged ions via gas-phase ion/ion reactions using weakly coordinating anions.

Selective removal of alkali metal cations from mixed cation multiply-charged peptide ions is demonstrated here using gas-phase ion/ion reactions with a series of weakly coordinating anions (WCAs), including hexafluorophosphate (PF6 (-)), tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (BARF), tetrakis(pentafluorophenyl)borate (TPPB), and carborane (CHB11Cl11 (-)). In all cases, a long-lived complex is generated by dication/anion condensation followed by ion activation to compare proton transfer with alkali ion transfer from the peptide to the anion. The carborane anion was the only anion studied to undergo dissociation exclusively through loss of the metallated anion, regardless of the studied metal adduct. All other anions studied yield varying abundances of protonated and metallated peptide depending on the peptide sequence and the metal identity. Density functional theory calculations suggest that for the WCAs studied, metal ion transfer is most strongly favored thermodynamically, which is consistent with the experimental results. The carborane anion is demonstrated to be a robust reagent for the selective removal of alkali metal cations from peptide cations with mixtures of excess protons and metal cations.

Continuous flow atmospheric pressure laser desorption/ionization using a 6-7-µm-band mid-infrared tunable laser for biomolecular mass spectrometry.

A continuous flow atmospheric pressure laser desorption/ionization technique using a porous stainless steel probe and a 6–7-µm-band mid-infrared tunable laser was developed. This ion source is capable of direct ionization from a continuous flow with a high temporal stability. The 6–7-µm wavelength region corresponds to the characteristic absorption bands of various molecular vibration modes, including O–H, C=O, CH3 and C–N bonds. Consequently, many organic compounds and solvents, including water, have characteristic absorption peaks in this region. This ion source requires no additional matrix, and utilizes water or acetonitrile as the solvent matrix at several absorption peak wavelengths (6.05 and 7.27 µm, respectively). The distribution of multiply-charged peptide ions is extremely sensitive to the temperature of the heated capillary, which is the inlet of the mass spectrometer. This ionization technique has potential for the interface of liquid chromatography/mass spectrometry (LC/MS).

Radical-driven peptide backbone dissociation tandem mass spectrometry.

In recent years, a number of novel tandem mass spectrometry approaches utilizing radical-driven peptide gas-phase fragmentation chemistry have been developed. These approaches show a peptide fragmentation pattern quite different from that of collision-induced dissociation (CID). The peptide fragmentation features of these approaches share some in common with electron capture dissociation (ECD) or electron transfer dissociation (ETD) without the use of sophisticated equipment such as a Fourier-transform mass spectrometer. For example, Siu and coworkers showed that CID of transition metal (ligand)-peptide ternary complexes led to the formation of peptide radical ions through dissociative electron transfer (Chu et al., 2000. J Phys Chem B 104:3393-3397). The subsequent collisional activation of the generated radical ions resulted in a number of characteristic product ions, including a, c, x, z-type fragments and notable side-chain losses. Another example is the free radical initiated peptide sequencing (FRIPS) approach, in which Porter et al. and Beauchamp et al. independently introduced a free radical initiator to the primary amine group of the lysine side chain or N-terminus of peptides (Masterson et al., 2004. J Am Chem Soc 126:720-721; Hodyss et al., 2005 J Am Chem Soc 127: 12436-12437). Photodetachment of gaseous multiply charged peptide anions (Joly et al., 2008. J Am Chem Soc 130:13832-13833) and UV photodissociation of photolabile radical precursors including a C-I bond (Ly & Julian, 2008. J Am Chem Soc 130:351-358; Ly & Julian, 2009. J Am Soc Mass Spectrom 20:1148-1158) also provide another route to generate radical ions. In this review, we provide a brief summary of recent results obtained through the radical-driven peptide backbone dissociation tandem mass spectrometry approach.

Activated Ion Negative Electron Transfer Dissociation of Multiply Charged Peptide Anions

We report the implementation and evaluation of activated ion negative electron transfer dissociation (AI-NETD) in order to enhance the analytical capabilities of NETD for the elucidation of doubly deprotonated peptide anions. The analytical figures-of-merit and fragmentation characteristics are compared for NETD alone and with supplemental collisional activation of the charge reduced precursors or infrared photoactivation of the entire ion population during the NETD reaction period. The addition of supplemental collisional activation of charge reduced precursor ions or infrared photoactivation of the entire ion population concomitant with the NETD reaction period significantly improves sequencing capabilities for peptide anions as evidenced by the greater abundances of product ions and overall sequence coverage. Neither of these two AI-NETD methods significantly alters the net fragmentation efficiencies relative to NETD; however, the sequence ion conversion percentages with respect to formation of diagnostic product ions are notably higher. Supplemental infrared photoactivation outperforms collisional activation for most of the peptide fragmentation metrics evaluated.

Liquid AP‐UV‐MALDI Enables Stable Ion Yields of Multiply Charged Peptide and Protein Ions for Sensitive Analysis by Mass Spectrometry

All charged up: High and prolonged yields of multiply charged peptide and protein ions can be formed in MALDI mass spectrometry using liquid UV-MALDI matrices and a heated ion-transfer tube. The key features are low laser energies of 1–10 μJ, resulting in fluences of less than 200 to 2000 J m−2 and low sample ablation, high sensitivity, and continuous ion generation over tens of thousands of laser shots.

High-definition differential ion mobility spectrometry with resolving power up to 500.

As the resolution of analytical methods improves, further progress tends to be increasingly limited by instrumental parameter instabilities that were previously inconsequential. This is now the case with differential ion mobility spectrometry (FAIMS), where fluctuations of the voltages and gas pressure have become critical. A new high-definition generator for FAIMS compensation voltage reported here provides a stable and accurate output than can be scanned with negligible steps. This reduces the spectral drift and peak width, thus improving the resolving power (R) and resolution. The gain for multiply-charged peptides that have narrowest peaks is up to ~40%, and R ~400-500 is achievable using He/N(2) or H(2)/N(2) gas mixtures.

Selective Isolation of Multiply Charged Peptides: A Confident Strategy for Protein Identification Using a Linear Trap Quadrupole Mass Spectrometer

In this work, a method devised for the selective isolation of multiply-charged peptide applied to a complex protein mixture was evaluated for the first time using a mass spectrometer with low resolution (LTQ). In this procedure, all primary amino groups of tryptic peptides derived from human Liver tissue interstitial fluid (TIF) are blocked, restricting their positive charge, at acidic pH, to the presence of histidine and arginine residues. After strong cation exchange chromatography, multiply-charged peptides (#R+#H > 1) are retained in the column and separated with high selectivity from singly (#R+#H = 1) and neutral peptides (#R +#H = 0) which are collected together in the flow-through. Using Liquid chromatography electrospray ionization tandem mass spectrometry analysis the retained fraction displayed a 95% enrichment of multiply charged peptides while in the flow-through; only 4% of multiply-charged peptides were identified.

ECD-like peptide fragmentation at atmospheric pressure

Fragmentation of multiply charged peptide ions via interaction with products of gas discharge at atmospheric pressure conditions was studied using ion mobility separation – fragmentation cell – linear ion trap mass spectrometer. The observed fragmentation spectra mainly consisted of c-type ions that are specific to electron capture dissociation. Experiments with different gases flowing through the discharge and different discharge polarities suggested that fragmentation proceeds via capture of free electrons. Fragmentation of a model phosphorylated peptide using this technique produced c-type fragments with an intact phosphorylation group. High field asymmetric waveform ion mobility separation of a peptide mixture prior to the fragmentation cell demonstrated the feasibility of conducting MS/MS-like experiments at atmospheric pressure conditions.

Identification of multiply charged proteins and amino acid clusters by liquid nitrogen assisted spray ionization mass spectrometry

The development of liquid nitrogen assisted spray ionization mass spectrometry (LNASI MS) for the analysis of multiply charged proteins (insulin, ubiquitin, cytochrome c, α-lactalbumin, myoglobin and BSA), peptides (glutathione, HW6, angiotensin-II and valinomycin) and amino acid (arginine) clusters is described. The charged droplets are formed by liquid nitrogen assisted sample spray through a stainless steel nebulizer and transported into mass analyzer for the identification of multiply charged protein ions. The effects of acids and modifier volumes for the efficient ionization of the above analytes in LNASI MS were carefully investigated. Multiply charged proteins and amino acid clusters were effectively identified by LNASI MS. The present approach can effectively detect the multiply charged states of cytochrome c at 400nM. A comparison between LNASI and ESI, CSI, SSI and V-EASI methods on instrumental conditions, applied temperature and observed charge states for the multiply charged proteins, shows that the LNASI method produces the good quality spectra of amino acid clusters at ambient conditions without applied any electric field and heat. To date, we believe that the LNASI method is the most simple, low cost and provided an alternative paradigm for production of multiply charged ions by LNASI MS, just as ESI-like ions yet no need for applying any electrical field and it could be operated at low temperature for generation of highly charged protein/peptide ions.

A New Model for Multiply Charged Adduct Formation Between Peptides and Anions in Electrospray Mass Spectrometry

A new model has been developed to account for adduct formation on multiply charged peptides observed in negative ion electrospray mass spectrometry. To obtain a stable adduct, the model necessitates an approximate matching of apparent gas-phase basicity (GB(app)) of a given proton bearing site on the peptide with the gas-phase basicity (GB) of the anion attaching at that site. Evidence supporting the model is derived from the fact that for [Glu] Fibrinopeptide B, higher GB anions dominated in adducts observed at higher negative charge states, whereas lower GB anions appeared predominately in lower charge state adducts. Singly charged adducts were only observed for lower GB anions: HSO(4)(-), I(-), CF(3)COO(-). Ions that have medium GBs (NO(3) (-), Br(-), H(2)PO(4)(-)) only form adducts having -2 charge states, whereas Cl(-) (higher GB) can form adducts having -3 charge states. The model portends that (1) carboxylate groups are much more basic than available amino groups; (2) apparent GBs of the various carboxylate groups on peptides do not vary substantially from one another; and (3) apparent GBs of the individual carboxylate and amino sites do not behave independently. This model was developed for negative ion attachment but an analogous mechanism is also proposed for the positive ion mode wherein (1) binding of a neutral at an amino site polarizes this amino group, but hardly affects apparent GBs of other sites; (2) proton addition (charge state augmentation) at one site can decrease the instrinsic GBs of other potential protonation sites and lower their apparent GBs.

Identification of Outer Membrane Proteins from an Antarctic Bacterium Pseudomonas syringae Lz4W

Subcellular fractionation of proteins is a preferred method of choice for detection and identification of proteins from complex mixtures such as bacterial cells. To characterize the membrane proteins of the Antarctic bacterium Pseudomonas syringae Lz4W, the membrane fractions were prepared using three different methods, namely Triton X-100 solubilization, sucrose density gradient, and carbonate extraction methods. The proteins were separated on one-dimensional polyacrylamide gels and analyzed using a combination of liquid chromatography-coupled electrospray ionization-MS. The membrane proteins that were prepared by carbonate extraction were separated on two-dimensional PAGE in different pI ranges using the detergent 2% amidosulfobetaine (ASB). The proteins were then subjected to matrix-assisted laser desorption ionization-time-of-flight/time-of-flight for analysis and identification. Because the genome sequence of P. syringae Lz4W is not known, the proteins were identified by using the relevant sequence databases of the Pseudomonas sp available at National Centre for Biotechnology Information (NCBI). The sequence identification of some tryptic peptides were validated by de novo sequencing and others by chemical modification and mass spectrometry. The peptide sequences of P. syringae Lz4W were then matched with the sequences of the peptides from different Pseudomonas sp. by similarity search of the proteins from different species using clustal W2 program. Thus by using a combination of the methods, we have been able to identify large number of proteins of this bacterial strain, which include most of the outer membrane proteins.

Commercial Intermediate Pressure MALDI Ion Mobility Spectrometry Mass Spectrometer Capable of Producing Highly Charged Laserspray Ionization Ions

The first examples of highly charged ions observed under intermediate pressure (IP) vacuum conditions are reported using laser ablation of matrix/analyte mixtures. The method and results are similar to those obtained at atmospheric pressure (AP) using laserspray ionization (LSI) and/or matrix assisted inlet ionization (MAII). Electrospray ionization (ESI), LSI, and MAII are methods operating at AP and have been shown, with or without the use of a voltage or a laser, to produce highly charged ions with very similar ion abundance and charge states. A commercial matrix-assisted laser desorption/ionization ion mobility spectrometry (IMS) mass spectrometry (MS) instrument (SYNAPT G2) was used for the IP developments. The necessary conditions for producing highly charged ions of peptides and small proteins at IP appear to be a pressure drop region and the use of suitable matrixes and laser fluence. Ionization to produce these highly charged ions under the low pressure conditions of IP does not require specific heating or a special inlet ion transfer region. However, under the current setup, ubiquitin is the highest molecular weight protein observed. These findings are in accord with the need to provide thermal energy in the pressure drop region, similar to LSI and MAII, to improve sensitivity and extend the types of compounds that produce highly charged ions. The practical utility of IP-LSI in combination with IMS-MS is demonstrated for the analysis of model mixtures composed of a lipid, peptides, and a protein. Further, endogenous multiply charged peptides are observed directly from delipified mouse brain tissue with drift time distributions that are nearly identical in appearance to those obtained from a synthesized neuropeptide standard analyzed by either LSI- or ESI-IMS-MS at AP. Efficient solvent-free gas-phase separation enabled by the IMS dimension separates the multiply charged peptides from lipids that remained on the delipified tissue. Lipid and peptide families are exceptionally well separated because of the ability of IP-LSI to produce multiple charging.