Comparison of electrospray and matrix-assisted laser desorption ionization on the same hybrid quadrupole time-of-flight tandem mass spectrometer: Application to bidimensional liquid chromatography of proteins from bovine milk fraction

Recently, two ionization sources, electrospray (ESI) and matrix-assisted laser desorption (MALDI) have been used in parallel to exploit their complementary nature and to increase proteome coverage. In this study, a method using bidimensional (2D) nanoLC coupled online with ESI quadrupole time-of-flight (Q-TOF) with the simultaneous collection of fractions for analyses by LC–MALDI Q-TOF–MS/MS was developed. A total of 39 bovine proteins were identified to a high degree of confidence. To help in differentiating peptide detection following ESI and MALDI with the same mass spectrometer, we compared physico–chemical characteristics of the peptides (molecular mass, charge and size) by principal component analysis (PCA) and analysis of variance on the results of PCA. More hydrophobic peptides with a wider mass coverage were identified when ESI was used, whereas more basic and smaller peptides were identified when MALDI was used. However, the generally accepted differentiation between ESI and MALDI according to the presence of basic amino acids residues Lys and Arg and the ratio Lys/Arg was not shown as significant in this study. Moreover, we pointed out the importance of the type of mass spectrometer used in complement to both ionization sources for achieving a global increase of proteome coverage.     

Decomposition of protein nitrosothiolsin matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry

The S-nitrosylation of proteins is involved in the trafficking of nitric oxide (NO) in intra- and extracellular milieus. To establish a mass spectrometric method for identifying this post-translational modification of proteins, a synthetic peptide and transthyretin were S-nitrosylated in vitro and analyzed by electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The intact molecular ion species of nitrosylated compounds was identified in the ESI mass spectrum without elimination of the NO group. However, the labile nature of the S-NO bond was evident when the in-source fragmentation efficiently generated [M + H - 30](+) ions. The decomposition was prominent for multiply charged transthyretin ions with high charge states under ordinary ESI conditions, indicating that the application of minimum nozzle potentials was essential for delineating the stoichiometry of nitrosylation in proteins. With MALDI, the S-NO bond cleavage occurred during the ionization process, and the subsequent reduction generated [M + H - 29](+) ions.     

Enhanced detection of phosphopeptides in matrix-assisted laser desorption/ionization mass spectrometry using ammonium salts

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has been used successfully to detect phosphorylation sites in proteins. Applications may be limited by the low response of phosphopeptides compared to nonphosphorylated peptides in MALDI MS. The addition of ammonium salts to the matrix/analyte solution substantially enhances the signal for phosphopeptides. In examples shown for equimolar mixtures, the phosphorylated peptide peaks become the largest peaks in the spectrum upon ammonium ion addition. This can allow for the identification of phosphopeptides in an unfractionated proteolytic digestion mixture. Sufficient numbers of protonated phosphopeptides can be generated such that they can be subjected to postsource decay analysis, in order to confirm the number of phosphate groups present. The approach works well with the common MALDI matrices such as α-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid, and with ammonium salts such as diammonium citrate and ammonium acetate.     

Stimulus Dependent Redistribution of Membrane Raft Cholesterol in Human Platelets

Cell membranes provide a requisite dynamic interface to facilitate communication between the extracellular environment and the intracellular milieu. These membranes contain proteins that span and/or are loosely associated with the lipid bilayer. The organization of lipids and proteins components into membrane micro-domains provides a temporal and spatial signaling platform for communication. Recently, cholesterol and sphingomyelin enriched membrane micro-domains known as lipid rafts have been implicated in cell signaling events. In these studies we have advanced our hypothesis that stimulus dependent rearrangement of cholesterol into and out of membrane rafts provides a unique lipid–mediated regulatory mechanism. Using fluorescent derivatives of cholesterol, we have shown that membrane raft associated cholesterol was altered in response to collagen-induced platelet aggregatory stimulation. Collagen stimulation resulted in a rapid redistribution of cholesterol from the outer to the inner membrane monolayer. The reorganization of the outer membrane monolayer resulted in a concomitant increase in outer monolayer fluidity. These studies are the first to show that membrane cholesterol was released from the exchangeable membrane raft pool in response to physiological stimuli.     

Analysis of a chlorosulfolipid from Ochromonas danica by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF) mass spectrometry (MS) is an established tool for analyzing high mass molecules, such as proteins, whereas it attracts far less interest in the field of lipid analysis. In the study reported here a new chlorosulfolipid (CSL), 3,8,12,15-tetrachloroeicosane-1,17,18-triyl tris(hydrogen sulfate), was identified from the alga Ochromonas danica and de novo characterized by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight (MALDI-QIT-ToF) MS in negative ion mode. This method provides an effective alternative for the analysis of compounds directly derived from organic cell extracts. For MALDI analyses several frequently used solid MALDI matrices as well as some ionic liquid matrices (ILMs) were tested to enhance the analyte response to UV-laser and its ionization. The molecular weight of the observed compound could be determined as Li-, Na- and K-adducts [M+Me-2H]-. The characteristic isotopic patterns of the measured ions and the well-allocated molecular fragments by MS1, MS2 and MS3 indicate the fourfold chlorination and threefold sulfation of the investigated compound. The MS fragmentation alongside of the chlorine-bearing C-atoms is accompanied by the generation of a double bond at the opposite fragment in MS1. This obtained fragmentation pattern provides an insight into the allocation of the chlorine-bearing C-atoms along the carbon chain.     

Matrix-assisted ionization vacuum for protein detection,fragmentation and PTM analysis on a high resolution linear ion trap-orbitrap platform

Matrix-assisted ionization vacuum (MAIV) is a novel ionization technique that generates multiply charged ions in vacuum without the use of laser ablation or high voltage. MAIV can be achieved in intermediate-vacuum and high-vacuum matrix-assisted laser desorption/ionization (MALDI) sources and electrospray ionization (ESI) sources without instrument modification. Herein, we adapt MAIV onto the MALDI-LTQ-Orbitrap XL platform for biomolecule analysis. As an attractive alternative to MALDI for in solution and in situ analysis of biomolecules, MAIV coupling to high resolution and accurate mass (HRAM) MS instrument has successfully expanded the mass detection range and improved the fragmentation efficiency due to the generation of multiply charged ions. Additionally, the softness of MAIV enables potential application in labile post-translational modification (PTM) analysis. In this study, proteins as large as 18.7 kDa were detected with up to 18 charges; intact peptides with labile PTM were well preserved during the ionization process and characterized MS/MS; peptides and proteins in complex tissue samples were detected and identified both in liquid extracts and in situ. Moreover, we demonstrated that this method facilitates MS/MS analysis with improved fragmentation efficiency compared to MALDI-MS/MS.     

Exploiting the complementary nature of LC/MALDI/MS/MS and LC/ESI/MS/MS for increased proteome coverage

The goal of this work was to evaluate the improvement in proteome coverage of complex protein mixtures gained by analyzing samples using both LC/ESI/MS/MS and LC/MALDI/MS/MS. Parallel analyses of a single sample were accomplished by interfacing a Probot fractionation system with a nanoscale LC system. The Probot was configured to perform a post-column split such that a fraction (20%) of the column effluent was sent for on-line LC/ESI/MS/MS data acquisition, and the majority of the sample (80%) was mixed with a matrix solution and deposited onto the MALDI target plate. The split-flow approach takes advantage of the concentration sensitive nature of ESI and provides sufficient quantity of sample for MALDI/MS/MS. Hybrid quadrupole time-of-flight mass spectrometers were used to acquire LC/ESI/MS/MS data and LC/MALDI/MS/MS data from a tryptic digest of a preparation of mammalian mitochondrial ribosomes. The mass spectrometers were configured to operate in a data dependent acquisition mode in which precursor ions observed in MS survey scans are automatically selected for interrogation by MS/MS. This type of acquisition scheme maximizes the number of peptide fragmentation spectra obtained and is commonly referred to as shotgun analysis. While a significant degree of overlap (63%) was observed between the proteins identified in the LC/ESI/MS/MS and LC/MALDI/MS/MS data sets, both unique peptides and unique proteins were observed by each method. These results demonstrate that improved proteome coverage can be obtained using a combination of these ionization techniques.     

Mass spectrometric analysis of chain end functionalization in ab initio cationic polymerization

Matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and LC-ESI MS was applied in the analysis of ab initio chain end functionalization in cationic polymerization of isobutyl vinyl ether. Well-resolved mass spectra of vinyl ether oligomers were obtained. The MALDI spectra give information on polymerization and functionalization process, as well as chain end functionality and side reactions occurred in the system.     

Multidimensional capillary array liquid chromatography and matrix-assisted laser desorption/ionization tandem mass spectrometry for high-throughput proteomic analysis

A two-dimensional capillary array liquid chromatography system coupled with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) was developed for high-throughput comprehensive proteomic analysis, in which one strong cation-exchange (SCX) capillary chromatographic column was used as the first separation dimension and 10 parallel reversed-phase liquid chromatographic (RPLC) capillary columns were used as the second separation dimension. A novel multi-channel interface was designed and fabricated for on-line coupling of the SCX to RPLC column array system. Besides the high resolution based on the combination of SCX and RPLC separation, the developed new system provided the most rapid two-dimensional liquid chromatography (2D-LC) separation. Ten three-way micro-splitter valves used as stop-and-flow switches in transferring SCX fractions onto RPLC columns. In addition, the three-way valves also acted as mixing chambers of RPLC effluent with matrix. The system enables on-line mixing of the LC array effluents with matrix solution during the elution and directly depositing the analyte/matrix mixtures on MALDI plates from the tenplexed channels in parallel through an array of capillary tips. With the novel system, thousands of peptides were well separated and deposited on MALDI plates only in 150min for a complex proteome sample. Compared with common 2D-LC system, the parallel 2D-LC system showed about 10-times faster analytical procedure. In combination with a high throughput tandem time of flight mass spectrometry, the system was proven to be very effective for proteome analysis by analyzing a complicated sample, soluble proteins extracted from a liver cancer tissue, in which over 1202 proteins were identified.     

How many proteins can be identified in a 2DE gel spot within an analysis of a complex human cancer tissue proteome?

Two‐dimensional gel electrophoresis (2DE) in proteomics is traditionally assumed to contain only one or two proteins in each 2DE spot. However, 2DE resolution is being complemented by the rapid development of high sensitivity mass spectrometers. Here we compared MALDI‐MS, LC‐Q‐TOF MS and LC‐Orbitrap Velos MS for the identification of proteins within one spot. With LC‐Orbitrap Velos MS each Coomassie Blue‐stained 2DE spot contained an average of at least 42 and 63 proteins/spot in an analysis of a human glioblastoma proteome and a human pituitary adenoma proteome, respectively, if a single gel spot was analyzed. If a pool of three matched gel spots was analyzed this number further increased up to an average of 230 and 118 proteins/spot for glioblastoma and pituitary adenoma proteome, respectively. Multiple proteins per spot confirm the necessity of isotopic labeling in large‐scale quantification of different protein species in a proteome. Furthermore, a protein abundance analysis revealed that most of the identified proteins in each analyzed 2DE spot were low‐abundance proteins. Many proteins were present in several of the analyzed spots showing the ability of 2DE‐MS to separate at the protein species level. Therefore, 2DE coupled with high‐sensitivity LC‐MS has a clearly higher sensitivity as expected until now to detect, identify and quantify low abundance proteins in a complex human proteome with an estimated resolution of about 500 000 protein species. This clearly exceeds the resolution power of bottom‐up LC‐MS investigations.     

Analysis of human plasma lipids and soybean lecithin by means of high‐performance thin‐layer chromatography and matrix‐assisted laser desorption/ionization mass spectrometry

An improved analytical strategy for the analysis of complex lipid mixtures using matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) in combination with high‐performance thin‐layer chromatography (HPTLC) is reported. Positive ion MALDI RTOF MS was applied as a rapid screening tool for the various neutral (e.g. triacylglycerols) and polar (e.g. glycerophospholipids and ‐sphingolipids) lipid classes derived from crude lipid extracts of e.g. human plasma as well as soybean lecithin. Finally, MALDI seamless post‐source decay (PSD) product ion analysis was performed in order to obtain further structural information (head‐ and acyl‐group identification) of selected lipid species and structure verification. A Coomassie Brilliant Blue R‐250 staining protocol for lipids on HPTLC plates was evaluated and was found to be fully compatible with subsequent MALDI‐MS. Lipids were analyzed after elution from the HPTLC phase material of the selected band (corresponding to certain lipid classes) by using the proper organic solvent mixture or in few cases directly from the HPTLC plates (a type of on‐line HPTLC/MALDI‐MS coupling). More than 70 distinct lipid species from seven different lipid classes in the range between m/z 500 and 1500 could be identified from the lipid extracts of human plasma and soybean lecithin, respectively. The general high sensitivity of MALDI‐MS detection allowed the analysis of even minor lipid classes from only very small volumes of human plasma (50 µL). The combination of HPTLC, Coomassie staining and positive ion MALDI curved field RTOF‐MS represents a straightforward strategy during lipidomics studies of food and clinically relevant human lipid samples. Copyright © 2009 John Wiley & Sons, Ltd.     

Detection of pharmaceutical compounds in tissue by matrix-assisted laser desorption/ionization and laser desorption/chemical ionization tandem mass spectrometry with a quadrupole ion trap

A novel quadrupole ion trap mass spectrometer laser microprobe instrument with an external ionization source was constructed and used to investigate the matrix-assisted laser desorption/ionization (MALDI) detection of pharmaceutical compounds in intact tissue. In addition to MALDI, laser desorption coupled with chemical ionization (LD/CI) was investigated. MALDI, using 2,5-dihydroxybenezoic acid (DHB) as a matrix, was employed to detect the anticancer drug paclitaxel from a thin section of rat liver tissue which had been incubated in a solution of paclitaxel. The results of that experiment showed that the ability to perform tandem mass spectrometry (MS/MS) with the quadrupole ion trap was crucial in the identification of drug compounds at trace levels in the complex tissue matrix. MALDI MS/MS was then used to detect the presence of paclitaxel in a human ovarian tumor at a concentration of approximately 50 mg/kg. Finally, the drug spiperone was detected in incubated rat liver tissue at an approximate level of 25 mg/kg using LD/CI (no MALDI matrix). Again, the MS/MS capability of the quadrupole ion trap was crucial in the identification of the drug at trace levels in the complex tissue matrix.     

Mass spectrometry imaging of triglycerides in biological tissues by laser desorption ionization from silicon nanopost arrays

Mass spectrometry imaging (MSI) is used increasingly to simultaneously detect a broad range of biomolecules while mapping their spatial distributions within biological tissue sections. Matrix‐assisted laser desorption ionization (MALDI) is recognized as the method‐of‐choice for MSI applications due in part to its broad molecular coverage. In spite of the remarkable advantages offered by MALDI, imaging of neutral lipids, such as triglycerides (TGs), from tissue has remained a significant challenge due to ion suppression of TGs by phospholipids, e.g. phosphatidylcholines (PCs). To help overcome this limitation, silicon nanopost array (NAPA) substrates were introduced to selectively ionize TGs from biological tissue sections. This matrix‐free laser desorption ionization (LDI) platform was previously shown to provide enhanced ionization of certain lipid classes, such as hexosylceramides (HexCers) and phosphatidylethanolamines (PEs) from mouse brain tissue. In this work, we present NAPA as an MSI platform offering enhanced ionization efficiency for TGs from biological tissues relative to MALDI, allowing it to serve as a complement to MALDI‐MSI. Analysis of a standard lipid mixture containing PC(18:1/18:1) and TG(16:0/16:0/16:0) by LDI from NAPA provided an ~49 and ~227‐fold higher signal for TG(16:0/16:0/16:0) relative to MALDI, when analyzed without and with the addition of a sodium acetate, respectively. In contrast, MALDI provided an ~757 and ~295‐fold higher signal for PC(18:1/18:1) compared with NAPA, without and with additional Na⁺. Averaged signal intensities for TGs from MSI of mouse lung and human skin tissues exhibited an ~105 and ~49‐fold increase, respectively, with LDI from NAPA compared with MALDI. With respect to PCs, MALDI provided an ~2 and ~19‐fold increase in signal intensity for mouse lung and human skin tissues, respectively, when compared with NAPA. The complementary coverage obtained by the two platforms demonstrates the utility of using both techniques to maximize the information obtained from lipid MS or MSI experiments.     

Proteomics annotation of lipid rafts modified by virus infection

Lipid rafts are sphingolipid- and cholesterol-enriched membrane microdomains, which are involved in entry, assembly and budding of various types of viruses. Identification of rafts associated proteins modified by virus infection could therefore provide novel insights into the mechanisms of virus infection as well as the development of new biomarkers for diagnosis and drug development. Proteomic approaches, such as LC-ESI-MS/MS, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), two-dimensional difference gel electrophoresis (2D-DIGE), isotope-coded affinity tags (ICAT), Proteolytic 1?O Labeling, isotope Tags for Relative and Absolute Quantification (iTRAQ), Stable Isotope Labeling with Amino acids in Cell culture (SILAC) and Multidimensional Protein Identification Technology (MudPIT), provide the large scale and unbiased platform to determine the dynamic profiles of the lipid rafts proteome. In this review, we summarized the research advance regarding modern proteomics analysis of host lipid rafts alterations in response to diverse virus infection.     

Improvements in protein identification confidence and proteome coverage for human liver proteome study by coupling a parallel mass spectrometry/mass spectrometry analysis with multi-dimensional chromatography separation

Recently, matrix-assisted laser desorption ionization (MALDI) technique has been shown to be complementary to electrospray ionization (ESI) with respect to the population of peptides and proteins that can be detected. In this study, we tried to hyphenate MALDI-TOF-TOF-MS and ESI-QUADRUPOLE-TOF-MS with a single 2D liquid chromatography for complicated protein sample analysis. The effluents of RPLC were split into two parts for the parallel MS/MS detection. After optimizing the operation conditions in LC separation and MS identification, a total of 1149 proteins were identified from the global lysate of normal human liver (NHL) tissue. Compared to the single MS/MS detection, the combined analysis increased the number of proteins identified (more than 25%) and enhanced the protein identification confidence. Proteins identified were categorized and analyzed based upon their cellular location, biological process and molecular function. The identification results demonstrated the application potential of a parallel MS/MS analysis coupled with multi-dimensional LC separation for complicated protein sample identification, especially for proteome analysis, such as human tissues or cells extracts.     

Crystalline hydration structure at the membrane-fluid interface of model lipid rafts indicates a highly reactive boundary region

The fluid mosaic model of biological membranes is that of a two-dimensional lipid bilayer in which both lipids and associated membrane proteins diffuse freely. More recently, the raft hypothesis proposed that membranes contain small, dynamic, functional domains (rafts), which act as platforms for membrane protein attachment and interaction. Although experimental evidence supporting the raft hypothesis is growing, very little is known of the structure of the membrane-fluid interface of lipid raft systems. Here, we report the direct submolecular-scale imaging of model raft membranes using ultrahigh resolution atomic force microscopy. We characterize the heterogeneous nature of crystalline hydration layers at the membrane-fluid interface. The association of crystalline hydration layers with raft membranes would significantly affect the mechanism and kinetics of both inter-raft interactions and those between rafts and external biomolecules, and therefore this finding has important implications for membrane biology.     

Chemically-assisted fragmentation combined with multi-dimensional liquid chromatography and matrix-assisted laser desorption/ionization post source decay, matrix-assisted laser desorption/ionization tandem time-of flight or matrix-assisted laser desorption/ionization tandem mass spectrometry for improved sequencing of tryptic peptides

Derivatization of tryptic peptides using an Ettan CAF matrix-assisted laser desorption/ionization (MALDI) sequencing kit in combination with MALDI-post source decay (PSD) is a fast, accurate and convenient way to obtain de novo or confirmative peptide sequencing data. CAF (chemically assisted fragmentation) is based on solid-phase derivatization using a new class of water stable sulfonation agents, which strongly improves PSD analysis and simplifies the interpretation of acquired spectra. The derivatization is performed on solid supports, ZipTip(microC18, limiting the maximum peptide amount to 5 microg. By performing the derivatization in solution enabled the labeling of tryptic peptides derived from 100 microg of protein. To increase the number of peptides that could be sequenced, derivatized peptides were purified using multidimensional liquid chromatography (MDLC) prior to MALDI sequencing. Following the first dimension strong cation exchange (SCX) chromatography step, modified peptides were separated using reversed-phase chromatography (RPC). During the SCX clean up step, positively charged peptides are retained on the column while properly CAF-derivatized peptides (uncharged) are not. A moderately complex tryptic digest, prepared from six different proteins of equimolar amounts, was CAF-derivatized and purified by MDLC. Fractions from the second dimension nano RPC step were automatically sampled and on-line dispensed to MALDI sample plates and analyzed using MALDI mass spectrometry fragmentation techniques. All proteins in the derivatized protein mixture digest were readily identified using MALDI-PSD or MALDI tandem mass spectrometry (MS/MS). More than 40 peptides were unambiguously sequenced, representing a seven-fold increase in the number of sequenced peptides in comparison to when the CAF-derivatized protein mix digest was analyzed directly (no MDLC-separation) using MALDI-PSD. In conclusion, MDLC purification of CAF-derivatized peptides significantly increases the success rate for de novo and confirmative sequencing using various MALDI fragmentation techniques. This new approach is not only applicable to single protein digests but also to more complex digests and could, thus, be an alternative to electrospray ionization MS/MS for peptide sequencing.     

Metabolite imaging using matrix-enhanced surface-assisted laser desorption/ionization mass spectrometry (ME-SALDI-MS)

We describe here the use of a hybrid ionization approach, matrix-enhanced surface-assisted laser desorption/ionization mass spectrometry (ME-SALDI-MS) in bioimaging. ME-SALDI combines the strengths of traditional matrix-assisted laser desorption/ionization (MALDI) and SALDI and enables successful MS imaging of low-mass species with improved detection sensitivity. Using 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) as the MS standard, MS performances of MALDI, SALDI, and ME-SALDI are systematically compared. The analyte desorption and ionization mechanism in ME-SALDI is qualitatively speculated based on the observation of significantly reduced matrix background and improved survival yields of molecular ions. Improvements in detection sensitivity of low-mass species using ME-SALDI over MALDI in imaging are demonstrated with mouse heart and brain tissues.     

The effect of sodium dodecyl sulfate and anion‐exchange silica gel on matrix‐assisted laser desorption/ionization mass spectrometric analysis of proteins

Sodium dodecyl sulfate (SDS), an anionic surfactant, is widely used in peptide and protein sample preparation. When the sample is analyzed by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS), this surfactant can often cause signal suppression. We have previously reported an on‐probe sample preparation method using a suspension of anion‐exchange silica gel and sinapinic acid (i.e., gel‐SA suspension) as a matrix, thereby greatly improving the MALDI signal detection of the protein solutions containing SDS. In this study, we found that a certain amount of SDS enhanced the MALDI signal intensity for protein samples. This effect was also observed when using sodium decyl sulfate and sodium tetradecyl sulfate instead of SDS. Furthermore, this on‐probe sample preparation method using both SDS and the gel‐SA suspension improved the detection limit of protein samples in the MALDI‐MS analysis by about ten‐fold as compared to that of protein samples without SDS and the gel‐SA suspension. This method can be applied not only to the MALDI‐MS analysis of samples containing SDS, but also to the examination of proteins at femtomole levels or insoluble proteins such as membrane proteins. Copyright © 2009 John Wiley & Sons, Ltd.     

Electrospray-assisted laser desorption/ionization and tandem mass spectrometry of peptides and proteins

We have constructed an electrospray-assisted laser desorption/ionization (ELDI) source which utilizes a nitrogen laser pulse to desorb intact molecules from matrix-containing sample solution droplets, followed by electrospray ionization (ESI) post-ionization. The ELDI source is coupled to a quadrupole ion trap mass spectrometer and allows sampling under ambient conditions. Preliminary data showed that ELDI produces ESI-like multiply charged peptides and proteins up to 29 kDa carbonic anhydrase and 66 kDa bovine albumin from single-protein solutions, as well as from complex digest mixtures. The generated multiply charged polypeptides enable efficient tandem mass spectrometric (MS/MS)-based peptide sequencing. ELDI-MS/MS of protein digests and small intact proteins was performed both by collisionally activated dissociation (CAD) and by nozzle-skimmer dissociation (NSD). ELDI-MS/MS may be a useful tool for protein sequencing analysis and top-down proteomics study, and may complement matrix-assisted laser desorption/ionization (MALDI)-based measurements.