小鼠晶体蛋白质组的双向电泳和质谱鉴定研究

目的应用双向电泳和质谱技术研究5周龄小鼠晶体蛋白质组。方法提取小鼠晶体总蛋白,进行固相pH梯度（IPG）等电聚焦双向电泳,胶体考马斯亮蓝R-250染色,使用PDQuest7．30图像分析软件分析电泳图像。选择主要蛋白点胶上酶解,应用基质辅助激光解析电离飞行时间／飞行时间（MALDI—TOF／TOF）仪器进行串联质谱（MS／MS）鉴定。结果上样量为882μg和190μg时,分别检测370±41蛋白点（n=3）和57±5个蛋白点（n=3）。高上样量能够较好地分离晶体低丰度蛋白,如念珠状纤维结构蛋白BFSP;低上样量可很好地分离高丰度蛋白-晶体蛋白（包括αA、αB;βA1～βA4;βB1～βB3;γA～γF和γS等）。质谱鉴定得到1种细胞骨架蛋白和16种高丰度晶体蛋白。结论双向电泳和质谱技术有效考察了晶体总蛋白质,为分析白内障形成过程中蛋白质的表达改变提供了新的方法和途径。     

Distinguishing Small Molecular Mass Differences of Proteins by Mass Spectrometry

Electrospray ionization–Fourier transform ion cyclotron resonance (ESI–FTICR) mass spectrometryallows for high-resolution, accurate mass analysisof multiply charged ions of proteins. In the workdescribed here, the ability of ESI–FTICR to distinguish small differences in molecular mass is evaluated. Ubiquitin was used as an internal mass calibration standard to measure the molecular mass of cytochromec, myoglobin, and several carbonic anhydrase isoforms. Mass calibration was based onthe tallest isotopic peak of each ubiquitin chargestate. Ubiquitin performed well as an internal standard because its charge states covered the appropriate mass range, interference was minimal, and thetallest peak was easily identified. The peak massesof cytochrome c (12.5 kDa) and myoglobin (17 kDa) were measured to an accuracy of about 0.02 Da (<2ppm). However, errors of 1.0 Da were observedfor some individual determinations because of the difficulty in identifying the tallest peak. When the technique was applied to bovine carbonic anhydrase II, even combining data from several charge statesdid not yield an unequivocal assignment of thetallest peak, resulting in a mass assignment of 29,023.7 or 29,024.7. Similarly, measurements of two isoforms with a mass difference of 1 Da, human carbonic anhydrase I, pI6.0 and 6.6, yielded overlapping values for the mass of the tallest peak. However, these two isoforms were clearly distinguished by (a) identification of the tallest peak using a measurement of average mass as a guide and (b) comparison of the isotopic peak intensity patterns.     

Effective Identification of Akt Interacting Proteins by Two-Step Chemical Crosslinking,Co-Immunoprecipitation and Mass Spectrometry

Akt is a critical protein for cell survival and known to interact with various proteins. However, Akt binding partners that modulate or regulate Akt activation have not been fully elucidated. Identification of Akt-interacting proteins has been customarily achieved by co-immunoprecipitation combined with western blot and/or MS analysis. An intrinsic problem of the method is loss of interacting proteins during procedures to remove non-specific proteins. Moreover, antibody contamination often interferes with the detection of less abundant proteins. Here, we developed a novel two-step chemical crosslinking strategy to overcome these problems which resulted in a dramatic improvement in identifying Akt interacting partners. Akt antibody was first immobilized on protein A/G beads using disuccinimidyl suberate and allowed to bind to cellular Akt along with its interacting proteins. Subsequently, dithiobis[succinimidylpropionate], a cleavable crosslinker, was introduced to produce stable complexes between Akt and binding partners prior to the SDS-PAGE and nanoLC-MS/MS analysis. This approach enabled identification of ten Akt partners from cell lysates containing as low as 1.5 mg proteins, including two new potential Akt interacting partners. None of these but one protein was detectable without crosslinking procedures. The present method provides a sensitive and effective tool to probe Akt-interacting proteins. This strategy should also prove useful for other protein interactions, particularly those involving less abundant or weakly associating partners.     

Multiplexed Quantitation of Endogenous Proteins in Dried Blood Spots by Multiple Reaction Monitoring - Mass Spectrometry

Dried blood spot (DBS) sampling, coupled with multiple reaction monitoring mass spectrometry (MRM-MS), is a well-established approach for quantifying a wide range of small molecule biomarkers and drugs. This sampling procedure is simpler and less-invasive than those required for traditional plasma or serum samples enabling collection by minimally trained personnel. Many analytes are stable in the DBS format without refrigeration, which reduces the cost and logistical challenges of sample collection in remote locations. These advantages make DBS sample collection desirable for advancing personalized medicine through population-wide biomarker screening. Here we expand this technology by demonstrating the first multiplexed method for the quantitation of endogenous proteins in DBS samples. A panel of 60 abundant proteins in human blood was targeted by monitoring proteotypic tryptic peptides and their stable isotope-labeled analogs by MRM. Linear calibration curves were obtained for 40 of the 65 peptide targets demonstrating multiple proteins can be quantitatively extracted from DBS collection cards. The method was also highly reproducible with a coefficient of variation of <15% for all 40 peptides. Overall, this assay quantified 37 proteins spanning a range of more than four orders of magnitude in concentration within a single 25 min LC/MRM-MS analysis. The protein abundances of the 33 proteins quantified in matching DBS and whole blood samples showed an excellent correlation, with a slope of 0.96 and an R² value of 0.97. Furthermore, the measured concentrations for 80% of the proteins were stable for at least 10 days when stored at −20 °C, 4 °C and 37 °C. This work represents an important first step in evaluating the integration of DBS sampling with highly-multiplexed MRM for quantitation of endogenous proteins.Dried Blood Spot (DBS)¹ samples have many advantages over blood serum or plasma and are the preferred clinical sample for newborn screening for metabolic diseases (1, 2). These samples are collected by pricking a newborn''s heel and spotting a drop of blood onto specially designed filter paper collection cards. Samples are then dried under ambient conditions and are usually stored with desiccant at room temperature until analysis. This sampling procedure is simpler and less invasive then intravenous blood draws, which require a trained phlebotomist. Not surprisingly, the majority of adult patients prefer the small lancet used in finger-prick blood sampling methods to the larger needles used in intravenous blood draws (3, 4). Unlike plasma or serum samples, which consume ≥250 μl of blood and must be centrifuged within an hour of collection, DBS samples can be prepared using a volume of only 10 μl, and do not require any specialized equipment at the collection site (5). The simplicity and reduced safety risks associated with DBS sampling enables collection by minimally trained staff or by the patients themselves. In addition, many analytes are stable in the DBS format at room temperature, reducing sample transportation and storage costs, as well as the impact on the environment. Finally, DBS samples are safer to transport and are considered exempt from dangerous goods regulations (6, 7). These advantages make DBS sampling very attractive for advancing personalized medicine and population-based biomarker research (8).Numerous biomolecular targets covering genomics, metabolomics, and proteomics applications have been quantified in DBS samples using a wide array of analytical techniques (9). The most common clinical application of DBS sampling is screening newborns for metabolomics disorders by targeting small molecule biomarkers. Early screening programs relied on bacterial inhibition assays and later immunoassays, both of which required a different assay for each target of interest (2). However, the time and cost required to perform each assay independently has limited the number of diseases that could be screened nationwide to only a handful. In addition, a single biomarker often lacked the specificity to produce a definitive diagnosis, requiring extensive secondary testing. Hemoglobin is the only protein that is commonly targeted in DBS samples, and primary screening is accomplished by high-performance liquid chromatography (HPLC) or isoelectric focusing (IEF) methods (2). Similar to small-molecule screening methods, these approaches are low-throughput and are not amendable to multiplexing with additional protein targets. In newborn screening programs, these challenges associated with small molecule analysis were overcome with the introduction of multiple reaction monitoring mass spectrometry (MRM-MS) into the clinical laboratories (1, 10). The specificity of MRM enables hundreds of analytes to be monitored during a single experiment to facilitate the development of highly multiplexed assays. The addition of stable isotope-labeled internal standards (SIS) enables the acquisition of highly reproducible results across a variety of instrumentation at different institutions. It is now common for 20–30 small molecule targets including amino acids, fatty acid acylcarnitines, and organic acid acylcarnitines to be analyzed by flow injection MRM-MS, at a cost of $10–20 USD per patient sample (11). Expansion of the screening panel to include additional small-molecule biomarkers on an existing platform may cost less than $1 each. In addition to newborn screening, DBS sampling combined with MS is also gaining acceptance in small-molecule drug development (12, 13). Here the collection of smaller blood volumes allows serial sampling from mice reducing the total number of animals required to generate preclinical toxicology and pharmacokinetic data (5).Despite the successful use of DBS samples in MS-based experiments for small molecule analysis, there have been few reports of using this technology for protein targets (13). Daniel and coworkers reported a screening method for identifying β-thalassemia using the well-known biomarker HbA₂, a hemoglobin variant composed of two alpha- and two delta-globin subunits (14). Proteins were extracted in an aqueous solution, digested with trypsin in 30 min and infused for MRM-MS analysis. Multiple hemoglobin peptides were targeted to measure the abundance of the delta-globin chain, using peptides from the beta-globin chain as an internal standard. This ratio correlated well with the abundance of intact HbA₂ as determined by a well-established HPLC method. The shorter acquisition time and the increased specificity of the MS-based method showed promise for improving population-wide screening. Boemer et al. used a similar flow injection MRM-MS strategy to screen newborns for hemoglobin variants associated with sickle cell disease (15). They analyzed more than 2000 DBS samples by targeting tryptic peptides that were unique to four different beta-globin mutations and compared their results with a standard IEF method that measured the intact proteins obtained from corresponding whole blood samples. Their flow injection MRM approach was able to identify the correct phenotype for all targeted variants. Recently, deWilde et al. reported a method for screening newborn DBS samples for ceruloplasmin, a protein linked to Wilson''s disease (16). Their method combined SIS peptides with LC/MRM-MS and produced results similar to those from an immunoassay for the analysis of seven patient samples. The monitoring of a therapeutic protein in rat blood was demonstrated with Kehler et al. to evaluate the suitability of this approach for supporting preclinical trials (17). Finally, a multiplexed approach was presented by Sleczka et al. for the simultaneous quantitation of two therapeutic proteins in spiked DBS samples collected from several animal models (18).In all previous methods, only one to two proteins were targeted in DBS samples and therefore the true multiplexing capabilities of MRM were not realized. MRM-based methods using SIS peptides have already proven proficient at highly multiplexed quantitation of proteins in plasma and serum samples (19–21). Our current work demonstrates the potential for integrating DBS sampling with LC/MRM-MS for highly multiplexed quantitation of endogenous proteins. Many of the 60 proteins that we have targeted have been cleared or approved by FDA, and are already being analyzed one at a time in clinical laboratories. The assay developed in this study includes a highly reproducible method for extracting multiple proteins from DBS samples followed by trypsin digestion and surrogate peptides, along with their SIS analogs, were analyzed by a standard-flow LC/MRM-MS platform that has previously been shown to give accurate, sensitive, and robust analysis of proteotypic peptides in human plasma (21, 22). The quantitative results from whole blood and the corresponding DBS samples were compared, and the integrity of DBS samples stored under various temperatures has been evaluated.     

Identification of Cell Cycle Dependent Interaction Partners of the Septins by Quantitative Mass Spectrometry

The septins are a conserved family of GTP-binding proteins that, in the baker''s yeast, assemble into a highly ordered array of filaments at the mother bud neck. These filaments undergo significant structural rearrangements during the cell cycle. We aimed at identifying key components that are involved in or regulate the transitions of the septins. By combining cell synchronization and quantitative affinity-purification mass-spectrometry, we performed a screen for specific interaction partners of the septins at three distinct stages of the cell cycle. A total of 83 interaction partners of the septins were assigned. Surprisingly, we detected DNA-interacting/nuclear proteins and proteins involved in ribosome biogenesis and protein synthesis predominantly present in alpha-factor arrested that do not display an assembled septin structure. Furthermore, two distinct sets of regulatory proteins that are specific for cells at S-phase with a stable septin collar or at mitosis with split septin rings were identified.Complementary methods like SPLIFF and immunoprecipitation allowed us to more exactly define the spatial and temporal characteristics of selected hits of the AP-MS screen.     

Easy Identification of Leishmania Species by Mass Spectrometry

Background

Cutaneous leishmaniasis is caused by several Leishmania species that are associated with variable outcomes before and after therapy. Optimal treatment decision is based on an accurate identification of the infecting species but current methods to type Leishmania isolates are relatively complex and/or slow. Therefore, the initial treatment decision is generally presumptive, the infecting species being suspected on epidemiological and clinical grounds. A simple method to type cultured isolates would facilitate disease management.

Methodology

We analyzed MALDI-TOF spectra of promastigote pellets from 46 strains cultured in monophasic medium, including 20 short-term cultured isolates from French travelers (19 with CL, 1 with VL). As per routine procedure, clinical isolates were analyzed in parallel with Multilocus Sequence Typing (MLST) at the National Reference Center for Leishmania.

Principal Findings

Automatic dendrogram analysis generated a classification of isolates consistent with reference determination of species based on MLST or hsp70 sequencing. A minute analysis of spectra based on a very simple, database-independent analysis of spectra based on the algorithm showed that the mutually exclusive presence of two pairs of peaks discriminated isolates considered by reference methods to belong either to the Viannia or Leishmania subgenus, and that within each subgenus presence or absence of a few peaks allowed discrimination to species complexes level.

Conclusions/Significance

Analysis of cultured Leishmania isolates using mass spectrometry allows a rapid and simple classification to the species complex level consistent with reference methods, a potentially useful method to guide treatment decision in patients with cutaneous leishmaniasis.     

PILOT_PROTEIN: Identification of Unmodified and Modified Proteins via High-Resolution Mass Spectrometry and Mixed-Integer Linear Optimization

A novel protein identification framework, PILOT_PROTEIN, has been developed to construct a comprehensive list of all unmodified proteins that are present in a living sample. It uses the peptide identification results from the PILOT_SEQUEL algorithm to initially determine all unmodified proteins within the sample. Using a rigorous biclustering approach that groups incorrect peptide sequences with other homologous sequences, the number of false positives reported is minimized. A sequence tag procedure is then incorporated along with the untargeted PTM identification algorithm, PILOT_PTM, to determine a list of all modification types and sites for each protein. The unmodified protein identification algorithm, PILOT_PROTEIN, is compared to the methods SEQUEST, InsPecT, X!Tandem, VEMS, and ProteinProspector using both prepared protein samples and a more complex chromatin digest. The algorithm demonstrates superior protein identification accuracy with a lower false positive rate. All materials are freely available to the scientific community at http://pumpd.princeton.edu .     

Rapid Identification of Clinical Yeast Isolates by Mass Spectrometry

Historically, yeasts and molds have been identified by their microscopy and macroscopy appearance, detection of specific antigens and metabolites, the results of biochemical testing, and the presence of species-specific gene sequences. These methods are either nonspecific or, in the case of gene sequencing, technically demanding, time-consuming, and expensive. Recent studies have demonstrated that matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) is a rapid, simple, inexpensive, and accurate tool for the identification of a wide spectrum of bacteria and fungi. There are also promising reports that this technology can be used for subtyping organisms and for performing antifungal susceptibility tests. This article reviews the applications of MALDI-TOF MS for routine clinical microbiology testing.     

Identification of Intact High Molecular Weight Glutenin Subunits from the Wheat Proteome Using Combined Liquid Chromatography-Electrospray Ionization Mass Spectrometry

The present paper describes a method for the identification of intact high molecular weight glutenin subunits (HMW-GS), the quality determining proteins from the wheat storage proteome. The method includes isolation of HMW-GS from wheat flour, further separation of HMW-GS by reversed-phase high-performance liquid chromatography (RP-HPLC), and their subsequent molecular identification with electrospray ionization mass spectrometry using a quadrupole-time-of-flight mass analyzer. For HMW-GS isolation, wheat proteins were reduced and extracted from flour with 50% 1-propanol containing 1% dithiothreitol. HMW-GS were then selectively precipitated from the protein mixture by adjusting the 1-propanol concentration to 60%. The composition of the precipitated proteins was first evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie staining and RP-HPLC with ultraviolet detection. Besides HMW-GS (≥65%), the isolated proteins mainly contained ω5-gliadins. Secondly, the isolated protein fraction was analyzed by liquid chromatography-mass spectrometry. Optimal chromatographic separation of HMW-GS from the other proteins in the isolated fraction was obtained when the mobile phase contained 0.1% trifluoroacetic acid as ion-pairing agent. Individual HMW-GS were then identified by determining their molecular masses from the high-resolution mass spectra and comparing these with theoretical masses calculated from amino acid sequences. Using formic acid instead of trifluoroacetic acid in the mobile phase increased protein peak intensities in the base peak mass chromatogram. This allowed the detection of even traces of other wheat proteins than HMW-GS in the isolated fraction, but the chromatographic separation was inferior with a major overlap between the elution ranges of HMW-GS and ω-gliadins. Overall, the described method allows a rapid assessment of wheat quality through the direct determination of the HMW-GS composition and offers a basis for further top-down proteomics of individual HMW-GS and the entire wheat glutenin fraction.     

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, ''hook-effect'').¹ An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).² In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules ^{3, 4} and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.^5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.^8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.     

Identification and Cluster Analysis of Streptococcus pyogenes by MALDI-TOF Mass Spectrometry

Background

Whole-cell matrix–assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has been successfully applied for bacterial identification and typing of many pathogens. The fast and reliable qualities of MALDI-TOF MS make it suitable for clinical diagnostics. MALDI-TOF MS for the identification and cluster analysis of Streptococcus pyogenes, however, has not been reported. The goal of our study was to evaluate this approach for the rapid identification and typing of S. pyogenes.

Methods

65 S. pyogenes isolates were obtained from the hospital. The samples were prepared and MALDI-TOF MS measurements were conducted as previously reported. Identification of unknown spectra was performed via a pattern recognition algorithm with a reference spectra and a dendrogram was constructed using the statistical toolbox in Matlab 7.1 integrated in the MALDI Biotyper 2.0 software.

Results

For identification, 61 of 65 S. pyogenes isolates could be identified correctly by MALDI-TOF MS with BioType 2.0 when compared to biochemical identification (API Strep), with an accuracy of 93.85%. In clustering analysis, 44 of 65 isolates were in accordance with those established by M typing, with a matching rate of 67.69%. When only the M type prevalence in China was considered, 41 of 45 isolates were in agreement with M typing, with a matching rate of 91.1%.

Conclusions

It was here shown that MALDI-TOF MS with Soft Biotype 2.0 and its database could facilitate rapid identification of S. pyogenes. It may present an attractive alternative to traditional biochemical methods of identification. However, for classification, more isolates and advances in the MALDI-TOF MS technology are needed to improve accuracy.     

Identification of Abscisic Acid in Tulipa gesneriana L. by Gas-Liquid Chromatography with Electron Capture and Combined Gas-Liquid Chromatography and Mass Spectrometry

A major growth inhibitory substance of tulip bulbs (Tulipa gesneriana L. cv Paul Richter) has been unequivocally shown to be abscisic acid (ABA). The ABA methyl ester of the free ether-soluble acid fractions of tulip organs had the identical retention time on gas-liquid chromatography with electron capture detector as authentic ABA methyl ester. In addition, the mass spectra were the same. On a unit dry matter basis, the basalplate and floral shoot contained 3.6 and 2.6 times more ABA than the fleshy scales, respectively.     

毛细管区带电泳／串联质谱联用法鉴定多肽和蛋白质

建立了毛细管区带电泳－串联质谱联用（ＣＺＥ／ＭＳ／ＭＳ）对多肽和蛋白质高灵敏度鉴定方法,对Ｍｅｔ－脑啡肽和Ｌｅｕ－脑啡肽的混合物进行了分析,用ＣＺＥ／ＭＳ／ＭＳ方法验证了各自的序列,同样对细胞色素ｃ的胰蛋白酶酶解产物用ＣＺＥ／ＭＳ／ＭＳ方法进行了肽质谱分析,几科所有肽段的序列及其与在分子中的位置都得到了确定,通过ＳＥＱＵＥＳＴ软件进行蛋白质序列数据库搜索得到准确的鉴定结果,所消耗的样品量均在低皮可     

Identification of Peptides Implicated in Antibacterial Activity of Snow Crab Hepatopancreas Hydrolysates by a Bioassay-Guided Fractionation Approach Combined with Mass Spectrometry

Snow crab (Chionoecetes opilio) by-products are a rich source of biomolecules, such as lipids, proteins, and chitin, which have not been extensively investigated. This study aims to identify antibacterial peptides to enhance the value of C. opilio by-products. After hydrolysis of different component parts using Protamex®, and concentration by solid-phase extraction, the resulting fractions were tested for antibacterial activity against Escherichia coli, Listeria innocua, and Vibrio parahaemolyticus. Hepatopancreas was the only tissue to display antibacterial activity detected using this protocol. Four fractions obtained with and without enzymatic hydrolysis of hepatopancreas followed by SPE C18 fractionation and elution with 50 and 80% acetonitrile demonstrated bacteriostatic activity against L. innocua HPB13, from concentrations of 0.30 to 43.05 mg/mL of peptides/proteins. Eleven peptides sharing at least 80% amino acid homology with four antimicrobial peptides were identified by mass spectrometry. Two peptides had homology to crustin-like and yellowfin tuna GAPDH antimicrobial peptides belonging to the marine organisms Penaeus monodon and Thunnus albacares, respectively. Other peptide sequence homologies were also identified: Odorranain-C7 from the frog Odorrana grahami and a predicted antibacterial peptide in the Asian ladybeetle Harmonia axyridis. These active peptides may represent a novel group of bioactive peptides deserving further investigation as food preservatives.

     

用于串联质谱鉴定多肽的计量方法

目前已有多种对串联质谱与数据库中多肽的理论质谱的一致性进行评估的高通量计量算法用于鸟枪法蛋白质组学 (shotgunproteomics)研究。然而这些方法操作时存在大量错误的多肽鉴定。这里提出一种新的串联质谱识别多肽序列的计量算法。该算法综合考虑了串联质谱中不同离子出现的概率、多肽的酶切位点数、理论离子与实验离子的匹配程度和匹配模式。对大容量的串联质谱数据集的测试表明 ,根据算法开发的软件PepSearch比目前最常用的软件SEQUEST有更好的鉴定准确性。PepSearch可从http : compbio.sibsnet.org projects pepsearch下载。     

Electron Capture Dissociation Mass Spectrometry in Characterization of Peptides and Proteins

Electron capture dissociation (ECD) represents one of the most recent and significant advancements in tandem mass spectrometry (MS/MS) for the identification and characterization of polypeptides. In comparison with the conventional fragmentation techniques, such as collisionally activated dissociation (CAD), ECD provides more extensive sequence fragments, while allowing the labile modifications to remain intact during backbone fragmentation—an important attribute for characterizing post-translational modifications. Herein, we present a brief overview of the ECD technique as well as selected applications in characterization of peptides and proteins. Case studies including characterization and localization of amino acid glycosylation, methionine oxidation, acylation, and “top–down” protein mass spectrometry using ECD will be presented. A recent technique, coined as electron transfer dissociation (ETD), will be also discussed briefly.