MALDI Tandem Mass Spectrometry Research Articles

Lipid A Structural Determination from a Single Colony.

We describe an innovative use for the recently reported fast lipid analysis technique (FLAT) that allows for the generation of MALDI tandem mass spectrometry data suitable for lipid A structure analysis directly from a single Gram-negative bacterial colony. We refer to this tandem MS version of FLAT as FLATn. Neither technique requires sophisticated sample preparation beyond the selection of a single bacterial colony, which significantly reduces overall analysis time (∼1 h), as compared to conventional methods. Moreover, the tandem mass spectra generated by FLATn provides comprehensive information on fragments of lipid A, for example, ester bonded acyl chain dissociations, cross-ring cleavages, and glycosidic bond dissociations, all of which allow the facile determination of novel lipid A structures or confirmation of expected structures. In addition to generating tandem mass spectra directly from single colonies, we also show that FLATn can be used to analyze lipid A structures taken directly from a complex biological clinical sample without the need for ex vivo growth. From a urine sample from a patient with an E. coli infection, FLATn identified the organism and demonstrated that this clinical isolate carried the mobile colistin resistance-1 gene (mcr-1) that results in the addition of a phosphoethanolamine moiety and subsequently resistance to the antimicrobial, colistin (polymyxin E). Moreover, FLATn allowed for the determination of the existence of a structural isomer in E. coli lipid A that had either a 1- or 4'-phosphate group modification by phosphoethanolamine generated by a change of bacterial culture conditions.

Ca2+ Complexation With Relevant Bioligands in Aqueous Solution: A Speciation Study With Implications for Biological Fluids.

A speciation study on the interaction between Ca2+ and ligands of biological interest in aqueous solution is reported. The ligands under study are l-cysteine (Cys), d-penicillamine (PSH), reduced glutathione (GSH), and oxidized glutathione (GSSG). From the elaboration of the potentiometric experimental data the most likely speciation patterns obtained are characterized by only protonated species with a 1:1 metal to ligand ratio. In detail, two species, CaLH2 and CaLH, for systems containing Cys, PSH, and GSH, and five species, CaLH5, CaLH4, CaLH3, CaLH2, and CaLH, for system containing GSSG, were observed. The potentiometric titrations were performed at different temperatures (15 ≤ t/°C ≤ 37, at I = 0.15 mol L−1). The enthalpy and entropy change values were calculated for all systems, and the dependence of the formation constants of the complex species on the temperature was evaluated. 1H NMR spectroscopy, MALDI mass spectrometry, and tandem mass spectrometry (MS/MS) investigations on Ca2+-ligand solutions were also employed, confirming the interactions and underlining characteristic complexing behaviors of Cys, PSH, GSH, and GSSG toward Ca2+. The results of the analysis of 1H NMR experimental data are in full agreement with potentiometric ones in terms of speciation models and stability constants of the species. MALDI mass spectrometry and tandem mass spectrometry (MS/MS) analyses confirm the formation of Ca2+-L complex species and elucidate the mechanism of interaction. On the basis of speciation models, simulations of species formation under conditions of some biological fluids were reported. The sequestering ability of Cys, PSH, GSH, and GSSG toward Ca2+ was evaluated under different conditions of pH and temperature and under physiological condition.

Site-Specific characterization of peptide-polymer conjugates in various stoichiometries by MALDI-Tandem mass spectrometry

PEGylation, covalent linking of polyethylene glycol (PEG) chains, is one of the most common ways for enhancing the drug properties of biopharmaceuticals. The linkage of PEG chains improves the pharmacokinetic properties of therapeutic biomolecules by increasing their hydrodynamic radius and also shields them against immune system. The chemical composition and structural features of biomolecule-PEG conjugates should be well-characterized in a fast and easy methods to project their therapeutic efficiency. For this purpose, mass spectrometry is the best analytical technique to analyze biopharmaceuticals in very low amounts with high sensitivity. Also, the stoichiometric ratio of species composing biomolecule-PEG conjugates can be easily calculated by using data obtained from mass spectrometric analysis. Moreover, tandem mass spectrometry (MS2) analysis, controlled breakdown of the isolated ions in the gas phase, also gives detailed information on chemical structures of species. The binding site of PEG chains on biomolecules can be determined by evaluating data obtained from MS2 analysis.In the study, angiotensin II peptide was PEGylated by using PEG chains with Mw=600 containing modified acid halide end-groups. MALDI-MS and MALDI-MS2 analyses were performed for obtaining data on determination of binding sites and stoichiometric ratio of angiotensin II and PEG chains for conjugates.

Abstract 389: Role of her3 signaling pathways in er+ and her2+ breast cancers

Abstract HER3 is an important molecule that may regulate oncogenic activity in estrogen receptor (ER)+ breast cancers, which accounts for about 70-80% of all breast cancers. HER3 gene expression is highest in ER+ or luminal tumors. Treatment with the ER degrader fulvestrant induces protein expression of HER3 in ER+ clinical breast tumors, suggesting that HER3 may have therapeutic value in luminal breast cancers. About 20% of all breast cancers diagnosed are HER2 positive (+). HER3 is as essential as HER2 for maintaining cell viability in HER2-overexpressing breast cancer cells. We aim to identify proteins that bind to HER3 upon therapeutic inhibition of HER2 or anti-endocrine therapy. We have found that ERα is upregulated in ER+ T47D and MCF-7 breast cancer cell lines expressing the activating hotspot T355I HER3 mutation compared to HER3 wild-type (WT) cells. We noted that ERα co-immunoprecipitated to HER3-T355I but not to HER3-WT T47D cells. Knocking down HER3 resulted in decreased ERα expression in both HER3-WT and HER3-T355I expressing T47D cells. We are currently analyzing the nuclear expression of endogenous HER3 in MCF-7, T47D, BT474, MDA-MB-361, MDA-MB-453 and SK-BR3 breast cancer cells. We are determining if stimulation with the HER3 ligand heregulin alters the subcellular location of HER3 or the ability to co-immunoprecipitate with ERα in cells expressing exogenous HER3-WT, exogenous HER3-T355I or endogenous HER3. We are using a proteomics approach to identify HER3-interacting proteins to determine how HER2 inhibition affects the repertoire of HER3 binding partners in HER2+ breast cancer cells. We immunopurified HER3 from BT474 cells treated with DMSO or with neratinib (24 hour treatment). We then identified enrichment of specific proteins by mass spectrometry. MALDI tandem mass spectrometry experiments identified non-muscle myosin-II heavy chain from HER3 immunoprecipitates treated 24 hours with neratinib treatment but not 24 hours of DMSO treatment in BT474 cells. Furthermore, our immunoprecipitation data indicated that there is more myosin-IIa bound to HER3 in response to neratinib treatment. Myosin-IIa has a key role in cytokinesis, cell migration, polarization, adhesion and metastasis. Thus, we aim to decipher the mechanism of action by which myosin-IIa interacts with HER3 and determining if downstream myosin/actin signaling could be a possible target in HER2 over-expressing breast cancer patients who show resistance to HER2 targeted therapy. In conclusion, we have identified two potential novel HER3 binding partners in breast cancer: ERα and myosin-IIa. Deciphering the function of HER3 binding partners has potential implication for understanding the invasive properties of ER+ and HER2+ breast cancers. Citation Format: Rosalin Mishra, Samar Alanazi, Hima Patel, Long Yuan, Joan T. Garrett. Role of her3 signaling pathways in er+ and her2+ breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 389.

Analysis of monodisperse, sequence-defined, and POSS-functionalized polyester copolymers by MALDI tandem mass spectrometry.

Monodisperse, sequence-defined polymers can be potentially used for digital data storage. This study reports the sequence analysis and differentiation of monodisperse polyester copolymers carrying side chains functionalized in a specific order by polyhedral oligomeric silsesquioxane (POSS) nanoparticles. Steglich esterification and succinic anhydride ring-opening chemistries were utilized iteratively to synthesize the intended sequences, which were characterized by matrix-assisted laser desorption ionization tandem mass spectrometry (MALDI-MS2). Isomeric oligomers were readily distinguished based on their different fragmentation patterns. The sequences embedded in the oligomers were decrypted by their specific backbone dissociation pathways. The robustness of using MALDI-MS2 as a sequencing method for monodisperse synthetic macromolecules was assessed and validated by the characterization of longer oligomers.

Matrix-assisted laser desorption/ionization tandem mass spectrometry for identification of organic tattoo pigments in inks and tissue samples.

With regard to the increasing number of tattooed people, legal regulations for tattoo inks were implemented across Europe-aiming for higher consumer safety. To control the laws' abidance, analytical methods are needed to identify banned ingredients from the given negative lists. Since specific organic pigments are often associated with tattoo side effects, their identification in tattoo inks as well as in biological samples is of great importance. Particularly, poorly soluble organic pigments are challenging to detect. In the past, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was reported as a promising tool for organic pigment identification. Here, we present a MALDI tandem mass spectrometry (MS/MS) approach to increase identification specificity and sensitivity in the process based on pigment fragment ions which is of special importance in tissue samples. For further verification of pigment identities, alkali metal cation attachment was used. Sample preparation was optimized and included mechanical disruption followed by the application as dried droplets with the matrices α-cyano-4-phenylcinnamic acid and sinapinic acid as well as ethanol. Pigments were identified by spectral comparison to reference libraries containing 40 pigments and following a decision tree. Additionally, successful pigment identification in biological samples was carried out. The implemented automated MALDI-MS and -MS/MS acquisitions make the hereby proposed pigment identification suitable for routine application.

Identification of α-enolase as a prognostic and diagnostic precancer biomarker in oral submucous fibrosis

AimsDiagnostic ambiguities regarding the malignant potentiality of oral submucous fibrosis (OSF), an oral precancerous condition having dysplastic and non-dysplastic isoforms are the major failure for early intervention of oral squamous...

Scrambled Internal Standard Method for High-Throughput Protein Quantification by Matrix-Assisted Laser Desorption Ionization Tandem Mass Spectrometry.

Matrix-assisted laser desorption ionization (MALDI) could be advantageous for high-throughput MS acquisition but suffers from low signal reproducibility. The purpose of this study was to establish a reliable MALDI-tandem mass spectrometry (MS/MS)-based high-throughput quantification of tryptic peptides using our newly developed scrambled internal standard (sIS) method. The standard curves obtained with sIS peptides showed good linearity over a wide concentration range (5-1000 fmol/μL) compared to that with the IS-free method, and the coefficient of variation of data points at each concentration (5-1000 fmol/μL) was significantly reduced. Furthermore, the ion suppression effect of digested serum could be normalized with the sIS peptides. Differences of quantitative values obtained by MALDI-MS/MS and liquid chromatography-MS/MS with selected reaction monitoring were within 20% in the presence of 0.1-5 μL of immunoprecipitated model plasma. Furthermore, the effect of amino acid composition on peptide sensitivity was examined, and we found that sensitivity was significantly decreased if an aromatic amino acid was replaced with a nonaromatic amino acid. Thus, high sensitivity required the use of sIS peptides containing an aromatic amino acid. Finally, the sIS method enabled high-throughput quantification of tryptic peptides with high accuracy and a wide dynamic range.

Single-cell MALDI Tandem Mass Spectrometry: Unambiguous Assignment of Small Biomolecules from Single Chlamydomonas reinhardtii Cells.

The analysis of compounds from single cells is a major challenge in analytical life science. Labeling strategies, for instance fluorescence detection, are well established for measuring proteins with single cell sensitivity, but they mostly fail to detect small molecules. More recently mass spectrometry has entered the realm of single cell sensitivity and enables the label-free and highly parallelized detection of small biomolecules from single cells. The assignment of signals detected in single cells, however, generally has to rely on measurements in whole cell culture extracts. Isobaric structures, contaminations, higher noise levels and the high variability in the abundance of peaks between single cells complicate the assignment of peaks in single-cell spectra. Tandem mass spectrometry would be very useful for compound identification via mass spectrometry directly in single-cell analyses. Here we present the first single cell tandem mass spectra collected using matrix-assisted laser-desorption/ionization. The spectra obtained allow the assignment of most compounds detected in the spectra. We also show that the fragmentation is not restricted to the most abundant peaks in the spectra, but over a dynamic range of more than one order of magnitude.

Characterization of immunoglobulins through analysis of N-glycopeptides by MALDI-TOF MS

The aim of this report is to emphasize the role, usefulness and power of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in the analysis of glycoforms of antibodies (Abs) through their proteolytic glycopeptides. Abs are complex biomolecules in which glycans hold determinant properties and thus need to be thoroughly characterized following Ab production by recombinant methods or Ab collection from human/animal serum or tissue. In spite of the great robustness of MALDI-TOF MS in terms of tolerance to impurities, the analysis of Abs and Ab components using this technique requires extensive sample preparation involving all or some of chromatography, solid phase extraction, enzymatic modification, and chemical derivatization. This report focuses on a monoclonal Ab produced in cell culture, as well as on a polyclonal human immunoglobulin (Ig) G obtained commercially and a polyclonal porcine IgG obtained from serum. A method is first provided to separate Ab protein chain components (light chains, heavy chains) by gel electrophoresis, which is useful for instance for protein-A eluates of Igs either from cell culture or biological samples. This allows for in-gel proteolytic digestion of the protein gel band(s) of choice for further MS characterization. Also discussed is the more conventional in-solution overnight digestion method used here with each of two proteolytic enzymes, i.e. trypsin and chymotrypsin. The overnight method is in turn compared with a much faster approach, that of digesting Abs with trypsin or chymotrypsin through the action of microwave heating. For method comparison, glycopeptides are fractionated from digestion mixtures using mostly C-18 cartridges for simplicity, although this enrichment procedure is also compared with other published procedures. The advantages of MALDI tandem mass spectrometry are highlighted for glycopeptide analysis, and lastly an esterification method applied to glycopeptides is discussed for retention of sialic acid residues on peptide acidic glycoforms.

A lipidomic approach to the study of human CD4(+) T lymphocytes in multiple sclerosis.

BackgroundLipids play different important roles in central nervous system so that dysregulation of lipid pathways has been implicated in a growing number of neurodegenerative disorders including multiple sclerosis (MS). MS is the most prevalent autoimmune disorder of the central nervous system, with neurological symptoms caused by inflammation and demyelination. In this study, a lipidomic analysis was performed for the rapid profile of CD4+ T lymphocytes from MS patient and control samples in an untargeted approach.MethodsA matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry based approach was used for the analysis of lipid extracts using 9-aminoacridine as matrix. Lipids were analyzed in negative mode and selected species fragmented using MALDI tandem mass spectrometry for their structural assignments.ResultsThe analysis reveals some modifications in the phospholipid pattern of MS CD4+ T lymphocytes with respect to healthy controls with a significant increase of cardiolipin species in MS samples.ConclusionsThese results demonstrate the feasibility of a MALDI-TOF approach for the analysis of CD4+ lipid extracts and suggest how alterations in the lipid metabolism characterized lymphocytes of MS patients.

The development of organometallic OBOC peptide libraries and sequencing of N-terminal rhenium(I) tricarbonyl-containing peptides utilizing MALDI tandem mass spectrometry

The development of peptide-based imaging agents through screening of large peptide libraries is hindered by the additional requirement of a radionuclide−chelator complex that can negatively affect the binding properties of the peptide. Herein, we report N-terminal rhenium(I)tricarbonyl OBOC (one-bead, one-compound) peptide libraries for use in the direct screening of potential imaging agents. The rhenium(I) tricarbonyl is incorporated directly in the library as an imaging entity surrogate to account for the presence of a technetium-99m radionuclide chelate. The identification of unknown organometallic peptides on single beads is successfully accomplished through MALDI tandem mass spectrometry, preceded by a systematic investigation of the effects of a variety of N-terminal rhenium(I) tricarbonyl chelates on peptide fragmentation patterns.

Structure elucidation of new acetylated saponins, Lessoniosides A, B, C, D, and E, and non-acetylated saponins, Lessoniosides F and G, from the viscera of the sea cucumber Holothuria lessoni.

Sea cucumbers produce numerous compounds with a wide range of chemical structural diversity. Among these, saponins are the most diverse and include sulfated, non-sulfated, acetylated and methylated congeners with different aglycone and sugar moieties. In this study, MALDI and ESI tandem mass spectrometry, in the positive ion mode, were used to elucidate the structure of new saponins extracted from the viscera of H. lessoni. Fragmentation of the aglycone provided structural information on the presence of the acetyl group. The presence of the O-acetyl group was confirmed by observing the mass transition of 60 u corresponding to the loss of a molecule of acetic acid. Ion fingerprints from the glycosidic cleavage provided information on the mass of the aglycone (core), and the sequence and type of monosaccharides that constitute the sugar moiety. The tandem mass spectra of the saponin precursor ions [M + Na]+ provided a wealth of detailed structural information on the glycosidic bond cleavages. As a result, and in conjunction with existing literature, we characterized the structure of five new acetylated saponins, Lessoniosides A–E, along with two non-acetylated saponins Lessoniosides F and G at m/z 1477.7, which are promising candidates for future drug development. The presented strategy allows a rapid, reliable and complete analysis of native saponins.

A major allergen in rainbow trout (Oncorhynchus mykiss): complete sequences of parvalbumin by MALDI tandem mass spectrometry

Fish parvalbumin (PRVB) is an abundant and stable protein in fish meat. The variation in cross-reactivity among individuals is well known and explained by a broad repertoire of molecular forms and differences between IgE-binding epitopes in fish species. PVRB has "sequential" epitopes, which retain their IgE-binding capacity and allergenicity also after heating and digestion using proteolytic enzymes. From the allergonomics perspective, PRVB is still a challenging target due to its multiple isoforms present at different degrees of distribution. Little information is available in the databases about PVRBs from Oncorhynchus mykiss. At present, only two validated, incomplete isoforms of this species are included in the protein databases: parvalbumin beta 1 (P86431) and parvalbumin beta 2 (P86432). A simple and rapid protocol has been developed for selective solubilization of PRVB from the muscle of farmed rainbow trout (Oncorhynchus mykiss), followed by calcium depletion, proteolytic digestion, MALDI MS, and MS/MS analysis. With this strategy thermal allergen release was assessed and PRVB1 (P86431), PRVB1.1, PRVB2 (P86432) and PRVB2.1 variants from the rainbow trout were sequenced. The correct ordering of peptide sequences was aided by mapping the overlapping enzymatic digests. The deduced peptide sequences were arranged and the theoretical molecular masses (Mr) of the resulting sequences were calculated. Experimental masses (Mr) of each PRVB variant were measured by linear MALDI-TOF.

Repeat MALDI MS imaging of a single tissue section using multiple matrices and tissue washes

Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) techniques are continually being assessed with a view to improving the quality of information obtained from a given sample. A single tissue section will typically only be analyzed once by MALDI MSI and is then either used for histological staining or discarded. In this study, we explore the idea of repeat analysis of a single tissue section by MALDI MSI as a route toward improving sensitivity, structural characterization, and diversity of detected analyte classes. Repeat analysis of a single tissue section from a fresh frozen mouse brain is investigated with both α-cyano-4-hydroxycinnamic acid (CHCA) and para-nitroaniline (PNA). Repeat analysis is then applied to the acquisition of MALDI MSI and MALDI tandem mass spectrometry imaging employing collision induced dissociation (MS/MS imaging employing CID) from a formalin-fixed mouse brain section. Finally, both lipid and protein data are acquired from the same tissue section via repeat analysis utilizing CHCA, sinapinic acid (SA), and a tissue wash step. PNA was found to outperform CHCA as a matrix for repeat analysis; multiple lipids were identified using MS/MS imaging; both lipid and protein images were successfully acquired from a single tissue section. Figure Repeat analysis by MALDI MS imaging of a single tissue section is investigated with multiple matrices and tissue washes to provide increased molecular information from a single tissue section.

ESI, APCI, and MALDI tandem mass spectrometry of poly(methyl acrylate)s: A comparison study for the structural characterization of polymers synthesized via CRP techniques and the software application to analyze MS/MS data

Research in polymer science and engineering is moving from classical methodologies to advanced analytical strategies in which mass spectrometry (MS)-based techniques play a crucial role. The molecular complexity of polymers requires new characterization tools and approaches to elucidate the detailed structural information. In this contribution, a comparison study of poly(methyl acrylate)s (PMA) using different tandem mass spectrometry techniques (ESI, APCI, and MALDI MS/MS) is reported to provide insights into the macromolecular structure with the aid of a special MS/MS data interpretation software. Collision-induced dissociation (CID) was utilized to examine the fragmentation pathways of PMAs synthesized via various controlled radical polymerization techniques. All three mass spectrometry techniques are used to analyze structural details of PMAs and the labile end-groups are determined based on the fragmentation behavior in CID. Fragmentation products were identified which are characteristics for the cleavage between the polymer chain and the end-group. The application of a tailor-made software is shown to analyze complex MS/MS data, and it is proven that this kind of software will be helpful for polymer scientists to identify fragmentation products obtained by tandem mass spectrometry similar to the fields of proteomics, metabolomics, genomics, and glycomics. © 2013 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013

Improved proteomic profiling of the cell surface of culture-expanded human bone marrow multipotent stromal cells

A comprehensive analysis of the membrane proteome is essential to explain the biology of multipotent stromal cells and identify reliable protein biomarkers for the isolation as well as tracking of cells during differentiation and maturation. However, proteomic analysis of membrane proteins is challenging and they are noticeably under-represented in numerous proteomic studies. Here we introduce new approach, which includes high pressure-assisted membrane protein extraction, protein fractionation by gel-eluted liquid fraction entrapment electrophoresis (GELFREE), and combined use of liquid chromatography MALDI and ESI tandem mass spectrometry. This report presents the first comprehensive proteomic analysis of membrane proteome of undifferentiated and culture-expanded human bone marrow multipotent stromal cells (hBM-MSC) obtained from different human donors. Gene ontology mapping using the Ingenuity Pathway Analysis and DAVID programs revealed the largest membrane proteomic dataset for hBM-MSC reported to date. Collectively, the new workflow enabled us to identify at least two-fold more membrane proteins compared to published results on hBM-MSC. A total of 84 CDs were identified including 14 CDs identified for the first time. This dataset can serve as a basis for further exploration of self-renewal, differentiation and characterization of hBM-MSC.

Ion/ion reactions of MALDI-derived peptide ions: increased sequence coverage via covalent and electrostatic modification upon charge inversion.

Atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI)-derived tryptic peptide ions have been subjected to ion/ion reactions with doubly deprotonated 4-formyl-1,3-benzenedisulfonic acid (FBDSA) in the gas-phase. The ion/ion reaction produces a negatively charged electrostatic complex composed of the peptide cation and reagent dianion, whereupon dehydration of the complex via collision-induced dissociation (CID) produces a Schiff base product anion. Collisional activation of modified lysine-terminated tryptic peptide anions is consistent with a covalent modification of unprotonated primary amines (i.e., N-terminus and ε-NH(2) of lysine). Modified arginine-terminated tryptic peptides have shown evidence of a covalent modification at the N-terminus and a noncovalent interaction with the arginine residue. The modified anions yield at least as much sequence information upon CID as the unmodified cations for the small tryptic peptides examined here and more sequence information for the large tryptic peptides. This study represents the first demonstration of gas-phase ion/ion reactions involving MALDI-derived ions. In this case, covalent and electrostatic modification charge inversion is shown to enhance MALDI tandem mass spectrometry of tryptic peptides.

Electron Detachment Dissociation of Underivatized Chloride-Adducted Oligosaccharides

Chloride anion attachment has previously been shown to aid determination of saccharide anomeric configuration and generation of linkage information in negative ion post-source decay MALDI tandem mass spectrometry. Here, we employ electron detachment dissociation (EDD) and collision activated dissociation (CAD) for the structural characterization of underivatized oligosaccharides bearing a chloride ion adduct. Both neutral and sialylated oligosaccharides are examined, including maltoheptaose, an asialo biantennary glycan (NA2), disialylacto-N-tetraose (DSLNT), and two LS tetrasaccharides (LSTa and LSTb). Gas-phase chloride-adducted species are generated by negative ion mode electrospray ionization. EDD and CAD spectra of chloride-adducted oligosaccharides are compared to the corresponding spectra for doubly deprotonated species not containing a chloride anion to assess the role of chloride adduction in the stimulation of alternative fragmentation pathways and altered charge locations allowing detection of additional product ions. In all cases, EDD of singly chloridated and singly deprotonated species resulted in an increase in observed cross-ring cleavages, which are essential to providing saccharide linkage information. Glycosidic cleavages also increased in EDD of chloride-adducted oligosaccharides to reveal complementary structural information compared to traditional (non-chloride-assisted) EDD and CAD. Results indicate that chloride adduction is of interest in alternative anion activation methods such as EDD for oligosaccharide structural characterization.

In situ Identification and Localization of IGHA2 in the Breast Tumor Microenvironment by Mass Spectrometry

Modifications in the tumor microenvironment (TME) play a major role in the establishment, progression, and metastasis of cancer. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a powerful technique that enables the simultaneous identification and localization of biological compounds within tissues. To detect markers of early TME remodeling in invasive breast cancer, we used MALDI-MSI to compare the molecular profiles of tissues from the breast cancer interface zone, tumor zone, and normal-tissue zone. Using direct-tissue MALDI tandem mass spectrometry (MS/MS), we identified immunoglobulin heavy constant alpha 2 (IGHA2) as a new, zone-specific protein in the breast TME. The zone-specific expression of IGHA2 was verified by immunoblotting and immunohistochemical analysis. IGHA2 expression was consistently positive in tumor cells that were metastatic to regional nodes, with intense expression along the cytoplasmic borders. As a factor related to an increased percentage of nodes with tumor metastasis, IGHA2 expression was upregulated 3.745-fold in cases with an increased number of cancerous nodes (p = 0.0468). Our results provide the first evidence of IGHA2 as a marker of the early process of TME remodeling in invasive breast cancer. Furthermore, IGHA2 may be a novel marker for regional metastases in the lymph nodes of patients with breast cancer.