MALDI Plate Research Articles

Streamlining regular liquid chromatography with MALDI-TOF MS and NMR spectroscopy using automatic full-contact splitless spotting interface and flash-tap fractioning collection

BackgroundHigh-resolution matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and nuclear magnetic resonance (NMR) spectroscopy are powerful tools to identify unknown psychoactive substances. However, in complex matrices, trace levels of unknown substances usually require additional fractionation and concentration. Specialized liquid chromatography systems are necessary for both techniques. The small flow rate of nano LC, typically paired with MALDI-TOF MS, often results in prolonged fractionation times. Conversely, the larger flow rate of semi-preparative LC, used for NMR analysis, can be time-consuming and labor-intensive when concentrating samples. To address these issues, we developed an integrated automatic system that integrated to regular LC. ResultAutomatic spot collector (ASC) and automatic fraction collector (AFC) were present in this study. The ASC utilized in-line matrix mixing, full-contact spotting and real time heating (50 °C), achieving great capacity of 5 μL droplet on MALDI plate, high recovery (76–116%) and rapid evaporation in 2 min. The analytes were concentrated 4–8 times, forming even crystallization, reaching the detection limit at the concentration of 50 μg L−1 for 12 psychoactive substances in urine. The AFC utilizes flexible tubing which flash-tapped the microtube's upper rim (3 mm depth) instead of reaching the bottom. This method prevents sample loss and minimizes the robotic arm's movement, providing a high fractionating speed at 6 s 12 psychoactive compounds were fractionated in a single round analysis (recovery: 81%–114%). Methamphetamine and nitrazepam obtained from drug-laced coffee samples were successful analyzed with photodiode array (PDA) after one AFC round and NMR after five rounds. SignificanceThe ASC device employed real-time heating, in-line matrix mixing, and full-contact spotting to facilitate the samples spotting onto the MALDI target plate, thereby enhancing detection sensitivity in low-concentration and complex samples. The AFC device utilized the novel flash-tapping method to achieve rapid fractionation and high recovery rate. These devices were assembled using commercially available components, making them affordable (400 USD) for most laboratories while still meeting the required performance for advanced commercialized systems.

Exploratory research on Neurogranin ratios for biomarkers of neurodegeneration

AbstractBackgroundNeurogranin (Ng) is a cerebrospinal fluid biomarker for synaptic dysfunction at early stage of Alzheimer’s disease (AD). It has been reported that some truncated forms from Ng is increased in AD brain tissue. However, the clinical significance of Ng forms in plasma has remained unclear. We have developed a simultaneous detection method of a plurality of Ng forms in plasma by using immunoprecipitation combined with matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (IP‐MALDI‐MS). In this study, we applied the IP‐MALDI‐MS technique to investigate blood‐based biomarkers for neurodegeneration.MethodPlasma samples were obtained from cognitively normal (CN), mild cognitive impairment (MCI), Alzheimer’s dementia (AD) and non‐Alzheimer’s dementia (non‐AD) individuals in National Center for Geriatrics and Gerontology. These subjects were classified as Aβ positive and negative based on PET with PIB tracer. Ng forms were immunoprecipitated from the samples spiked by stable isotope‐labeled (SIL) Ng peptides using magnetic beads coupled with anti‐Ng monoclonal antibody. After the IP was performed two times consecutively, Ng forms eluted from antibody beads were applied on the MALDI plate. SIL‐Ng peptides were used as internal standards for normalizing an intensity of each endogenous Ng form in plasma.ResultWe calculated intensity ratio of a Ng form‐to‐other Ng form (Ng ratio) in all combinations of peaks corresponding to mass of Ng forms. To investigate the possibility of Ng as a biomarker for neurodegeneration, we screened the Ng ratios with the difference between CN and Aβ positive AD or Aβ negative non‐AD. Eleven Ng ratios were significantly higher in the Aβ positive AD and Aβ negative non‐AD groups than the CN group. In ROC analyses, AUC values greater than 0.750 were found in discriminating the Aβ positive AD or Aβ negative non‐AD group from the CN group. Among the eleven Ng ratios, five were significantly correlated with MMSE (ρ←0.400).ConclusionSome Ng forms cleaved at or nearly the IQ domain have been found in the AD brain, which suggests the function loss of Ng in synaptic plasticity. Increasing Ng ratios in plasma may be biomarker candidates for synaptic dysfunction involved in the change of Ng forms in the brain.

B-371 Two Cases of Multiple Myeloma Achieving Complete Remission But Presenting Residual M-protein by the EXENT® Solution

Abstract Background Disappearance of the monoclonal component is a condition for achieving complete response (CR), which requires confirmation by bone marrow biopsy for the absence of clonal plasma cells. Electrophoretic methods used for monitoring these patients lack sufficient sensitivity to identify low-level M-protein, potentially missing the presence of serological residual disease once the tests become negative. The EXENT® solution (in development by The Binding Site, part of Thermo Fisher scientific) uses matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for the identification and quantification of M-proteins. The technique offers high sensitivity and specificity for identifying and tracking M-proteins. The aim of this study is to determine whether the superior analytical sensitivity of mass spectrometry conveys any clinical benefit to patient monitoring. Methods We present two multiple myeloma patients seen during routine practice. Baseline and longitudinal samples were available. Serum samples were retrospectively analysed with the EXENT® solution. Briefly, diluted samples were separately incubated with antisera specific for IgG, IgA, IgM, total-κ and total-λ conjugated to paramagnetic beads. Purified immunoglobulins were reduced and eluted from the beads, the solution mixed with matrix, spotted onto MALDI plates, and acquired by MALDI-TOF-MS to determine mass-to-charge (m/z), isotype and concentration of the M-proteins. Results were compared to serum protein electrophoresis (SPE) and free light chains (FLC; Freelite, The Binding Site). The EXENT solution responses were modelled on those from the International Myeloma Working Group. CR by MS refers to absence of the original monoclonal peak during follow-up. Results Case 1 is a 68-year-old white male presenting with 11.7 g/L monoclonal IgGκ on SPE and abnormal FLC ratio (3.06). After initiating therapy, FLC ratio normalized at month 2, whereas SPE became negative at month 8, at which time a CR was confirmed. The patient stayed in remission for over two-and-a-half years before relapsing at month 40, as determined by re-appearance of the M-protein on SPE (2.20 g/L). The EXENT solution also identified an IgGκ peak at baseline (m/z = 11629, 11.9 g/L). The peak remained present until month 19, disappeared for over a year, then reemerged at month 36 (0.39 g/L) to indicate progression. Thus, the EXENT solution identified the presence of residual M-protein (<1 g/L) for an additional eleven months after standard monitoring methods indicated CR; and recognized relapse from CR four months before SPE.Case 2 is a 76-year-old Asian male presenting with 32.9 g/L monoclonal IgGλ on SPE and normal FLC ratio (0.43). The EXENT solution reported an IgGλ peak (m/z = 11250, 44.3 g/L) at this time. SPE became negative ten months after starting treatment; and identified relapse from CR one year after. The EXENT solution remained positive throughout and was the only method detecting low-level M-protein (≤1.25 g/L) during the 1-year remission period. Conclusion EXENT demonstrates a potentially superior performance over electrophoretic methods for identifying residual serological disease during monitoring; and shows potential as a serum biomarker of early relapse in myeloma. The improved sensitivity of MS suggests it may delay and, in some cases, avoid the need for invasive methods to determine response to therapy.

Strategies for MALDI-MS method development to investigate different pharmaceutical drug modalities

High-throughput screening (HTS) is an ultra-high throughput tool that allows the identification of diverse, pharmaceutically active compounds in the early stages of drug discovery, by enabling chemists to screen reactions at micromole/nanomole scales. To eliminate the bottleneck arising from analysis of large libraries, Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) can be utilized as a compatible, ultra-fast, label-free, analysis tool. Upon integrating this technique with suitable liquid handling robots that can accurately spot matrices and analytes on a MALDI plate, chemical reactions can be screened at a sampling rate of <1 s per sample. While earlier efforts to improve MALDI-HTS applications were geared towards the development of automation and data processing tools, a systematic method development study across different drug modalities was needed to understand the contribution of different parameters such as MALDI matrices, choice of matrices, additives, and solvent composition, on MALDI-MS signal intensities of analytes. The current study will demonstrate the importance of initial MALDI-MS method development on small molecules, peptides, antibodies, and antibody drug conjugates. This study is designed as a starting point for MALDI-MS signal optimization, which can eventually lead to the development of effective MALDI-HTS methods for various modalities, thus paving the way for a wide range of applications, including but not limited to conformational analysis.

Miniaturized affinity chromatography: A powerful technique for the isolation of high affinity GAGs sequences prior to their identification by MALDI-TOF MS

Glycosaminoglycans (GAGS) are involved in many biological processes through interactions with a variety of proteins, including proteases, growth factors, cytokines, chemokines and adhesion molecules. Identifying druggable GAG-protein interactions for therapeutic purposes is a challenge for the analytical community. In this context, this work investigates the use of a new miniaturized monolithic affinity column (poly(GMA-co-MBA) grafted with antithrombin III (AT III)) to specifically capture and elute high affinity sequences contained in low molecular weight heparin (enoxaparin) for further on-line characterization. This miniaturized, high binding capacity affinity column allows the specific capture of high-affinity oligosaccharide chains from Enoxaparin, even at low concentrations and with a minimal consumption of AT III. In addition to purification, this elution process enables preconcentration for direct analysis by capillary zone electrophoresis. It was found that many of oligosaccharide chains in enoxaparin were eliminated and that certain chain sequences were retained and enriched. Direct coupling with MALDI-TOF MS was successfully used to further characterize the specifically retained oligosaccharides where nano-ESI-TOF MS failed. After optimization of the sample preparation and ionization parameters, direct on-line analysis was performed by applying the elution volume released from the miniaturized affinity column (≤1 μL) directly to the MALDI plate. Finally, this original miniaturized analytical workflow coupling miniaturized AT III-affinity chromatography to MALDI-TOF MS detection is able to select, enrich and detect and identify high affinity sequences (mainly DP4 in size length with a high degree of sulfation) from low molecular weight heparin samples. A more specific selection of GAG sequences can be achieved by increasing the ionic strength during the washing step of affinity chromatography. This is consistent with the known binding pattern between heparin and AT III.

Poly(L-dopa) film-assisted laser desorption/ionization of small biomolecules and drugs

Although matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) owns unique merits, the applicability of MALDI for small molecule analysis is still very limited due to the matrix-related interferences in low-mass region. We present a new strategy for the analysis of small biomolecules and drugs by LDI MS. Poly(L-dopa) film was in situ polymerized on standard stainless steel MALDI plate, and the analytes deposited on the film were vaporized and ionized under laser irradiation. Poly(L-dopa) film was found highly effective for laser desorption/ionization of small molecules including amino acids, organic acids, nucleosides, monosaccharides and sulfa drugs at pmol levels with extremely low background noise. Amino acids was detectable even in saturated salt solution. The good salt tolerance and high sensitivity allowed its application to the detection of celecoxib in serum at concentration as low as 0.29 μM. The preparation of poly(L-dopa) film is very simple and convenient. It is believed that poly(L-dopa) film-assisted laser desorption/ionization mass spectrometry has potential to be a practical solution for small biomolecule analysis.

The rapid detection of procalcitonin in septic serum using immunoaffinity MALDI chips

BackgroundSepsis is a common worldwide health condition with high mortality. It is caused by a dysregulated immune response to the pathogen. Severe infections resulting in sepsis can be also determined by monitoring several bloodstream biomarkers, one of them being pro-hormone procalcitonin (PCT). PCT concentration in the bloodstream correlates well with sepsis and in severe cases increases up to a thousand times from the healthy physiological values in a short time. In this study, we developed a rapid technique for PCT detection by MALDI-TOF mass spectrometry, that uses in-situ enrichment directly on the specialized immuno MALDI chips that are utilized as MALDI plates. The method’s ability to detect PCT was confirmed by comparing the results with LC–MS bottom-up workflow. The new method detects intact PCT by its m/z and uncovers its alternations in septic serum.MethodsThe MALDI chips used for the detection of PCT were prepared by ambient ion soft landing of anti-PCT antibody on an ITO glass slide. The chips were used for the development of the rapid MALDI-TOF MS method. A parallel method based on affinity enrichment on magnetic beads followed by LC–MS/MS data-dependent peptide microsequencing was used to prove PCT presence in the sample. All samples were also tested by ELISA to determine PCT concentration prior to analyzing them by mass spectrometry methods.ResultsThe MALDI chip method was optimized using recombinant PCT spiked into the human serum. The PCT detection limit was 10 ng/mL. The optimized method was used to analyze 13 sera from patients suffering sepsis. The PCT results were confirmed by LC–MS/MS. The measurement of the intact PCT by the MALDI chip method revealed that sera of patients with severe sepsis have other forms of PCT present, which show post-processing of the primary sequence by cleavage of PCT, resulting in the formation of N and C termini fragments.ConclusionsProcalcitonin from human serum was successfully enriched and detected using immunoaffinity MALDI chips. The intact PCT was characterized in 13 septic patients. The method is more specific compared to non-MS-based immunoaffinity techniques and allows observation of different variants of PCT in septic patients.

P879: EXENT AND FLC MASS SPECTROMETRY FOR SEROLOGICAL IDENTIFICATION AND QUANTIFICATION OF MONOCLONAL IMMUNOGLOBULINS IN MULTIPLE MYELOMA

Background: Mass spectrometry has broad potential for screening and monitoring monoclonal gammopathies. The technology overcomes some of the limitations inherent to standard electrophoretic methods for identifying monoclonal immunoglobulins and is amenable to automation. Aims: Here we assess the performance of the EXENT assays for the quantification of M-proteins and whether free light chain evaluation by mass spectrometry (FLC-MS) offers added sensitivity during multiple myeloma monitoring. Methods: The MM5 trial compared two regimens of bortezomib-based induction therapy and of lenalidomide consolidation followed by lenalidomide maintenance in newly diagnosed multiple myeloma patients. We used mass spectrometry for identifying serum M-proteins in samples at baseline (n=434), post-induction (n=340), and post-consolidation (n=294). Briefly, polyclonal antibodies (anti-IgG,-IgA,-IgM,-total κ and -total λ [EXENT, The Binding Site, UK] and anti-free κ and -free λ [FLC-MS]) covalently attached to paramagnetic microparticles were separately incubated with serum, washed, treated to elute and reduce patient immunoglobulins, and spotted onto MALDI plates with HCCA matrix. Spectra were generated using the MALDI-TOF-MS system. Light chain mass spectra were obtained using EXENT software to yield immunoglobulin isotype, mass-to-charge ratio (m/z) and quantity (g/L). Results were compared to serum protein electrophoresis, immunofixation, and FLC ratios (Freelite®, The Binding Site, UK). Results: EXENT demonstrated improved sensitivity over immunofixation for identifying the M-protein at each time-point of the MM5 trial: 99% vs. 95% at baseline; 91% vs 80% post-induction; and 63% vs. 46% post-consolidation. Agreement between methods was 87%. The addition of FLC-MS to EXENT further improved sensitivity to 100%, 95.0%, and 72% at each respective time-point, whereas the combination of immunofixation and FLC ratios resulted in sensitivities of 100%, 90%, and 54%. Overall concordance between EXENT+FLC-MS and the combination of immunofixation and FLC ratios was 100% at baseline, 93% post-induction, and 73% post-consolidation. In all, 158 (11%) samples had an M-protein by EXENT+FLC-MS only, and 28 (2%) by the combination of immunofixation and FLC ratios. 18 and 92 patients achieved an IMWG complete response after induction and consolidation, respectively. EXENT identified an M-protein in 5 (28%), and 23 (25%) of these patients, whereas EXENT+FLC-MS was more sensitive and detected monoclonal proteins in 11 (61%), and 38 (41%) patients, respectively. There was acceptable quantitative agreement for M-protein measurements between EXENT and serum protein electrophoresis (n=631: Passing-Bablok fit: y=0.85x-0.58; coefficient of determination R2= 0.89; Bland-Altman relative bias (95%CI): -32.62% (-123.79-58.55%)). However, there was a substantial discordance in 285 samples <10g/L by serum protein electrophoresis (y=0.58x+0.54; R2=0.28; -44.84% (-179.29 – 84.60%)) compared to 346 samples >10g/L (y=0.85x-0.25; R2=0.82; -19.91% (95%CI: -97.32 – 23.56%)); suggesting overestimation by serum protein electrophoresis at lower levels. Summary/Conclusion: EXENT provides superior performance for the identification of M-proteins throughout monitoring. Sensitivity can be improved by incorporating FLC-MS assessments, thus providing a means for determining serological residual disease in patients at complete response by IMWG criteria. Quantification of the M-protein by mass spectrometry compares well to standard methods and offers accurate results at levels below the analytical threshold of confidence for electrophoretic approaches.

Detection and characterization of simvastatin and its metabolites in rat tissues and biological fluids using MALDI high resolution mass spectrometry approach

Simvastatin (SV) is a hypolipidemic agent, and it is the 2nd most widely prescribed lipid-lowering drug. Here, the detection and characterization of SV and its metabolites was studied in selected organs/tissues (lung, liver, brain, heart and kidney) and biological samples (blood, urine and feces) of rats. MALDI Orbitrap MS was used as a high-resolution mass analyzer. 2,5-Dihydroxybenzoic acid (DHB) and 1,5-diaminonaphthalene (DAN) were used as matrices. Several sample loading methods onto the MALDI plate were attempted and dried droplet method was found to be superior. Two different cell disruption methods, pulverization and homogenization, were also evaluated for the optimum sensitivity in MALDI. Pulverization allowed the detection of more metabolites in all organs except the liver, where homogenization led to the detection of more metabolites. Altogether, 13 metabolites were detected, and one metabolite tentatively identified as a reduced product is being reported for the first time. SV and its metabolites were distributed to all the tissues studied except the brain. Overall, the results implied that the pulverized samples were more uniform and larger in surface area, resulting in their more efficient and complete extraction during sample preparation. As shown in the present study, MALDI Orbitrap MS is a useful tool to study drug and metabolite detection and characterization.

Detection of multiply charged protein ions using matrix-assisted laser desorption/ionization mass spectrometry and a force-dried droplet method with a 2-nitrophloroglucinol matrix.

Conventional dried droplet (DD) methods show poor reproducibility in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) due to the frequent induction of a heterogeneous sample distribution. Recently, a forced dried droplet (FDD) sample preparation method was introduced to form homogeneous samples; this method improves the reproducibility of MALDI-MS analysis and generates highly multiply charged ions compared to DD methods. The FDD method utilizes secondary nucleation to generate a homogeneous sample distribution by applying an external force such as fluid shear stress by stirring the sample using a micropipette tip. In this study, a 2-nitrophloroglucinol (2-NPG) matrix was used for the DD and FDD sample preparation methods, and the charge state and homogeneity were compared by detecting multiply charged ions of proteins including cytochrome c, myoglobin, and immunoglobulin G (IgG) and measuring the relative standard deviation (RSD). FDD with a 2-NPG matrix produced a more homogeneous sample distribution and higher charge state ions than the DD method. FDD with a 2-NPG matrix was applied in MALDI-MS analysis of IgG fragments obtained from sequential reduction of IgG. In addition, FDD with intentional scratching of the MALDI plate by rotating a micropipette tip was found to provide similar or better reproducibility, higher charge state ions, and more uniformly distributed sample morphology compared to FDD without scratching.

Semi‐quantitative assay for neurogranin peptides using IP‐MALDI‐MS

AbstractBackgroundNeurogranin (Ng) is a cerebrospinal fluid (CSF) biomarker for synaptic dysfunction at early stage of Alzheimer’s disease (AD). It has been reported that a variety of truncated peptides from Ng is increased in AD brain tissue. However, the clinical significance of Ng peptides in plasma has remained unclear. We have developed a simultaneous detection method of a plurality of amyloid βpeptides in plasma by using immunoprecipitation combined with matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (IP‐MALDI‐MS) (Kaneko et al., Proc Jpn Acad Ser B Phys Biol Sci. 2014). In this study, we applied the IP‐MALDI‐MS technique to Ng measurement.MethodNg peptides and proteins were immunoprecipitated from human plasma or standard sample spiked by stable isotope‐labeled (SIL) Ng50‐78 using magnetic beads coupled with anti‐Ng monoclonal antibody. After the IP was performed two times consecutively, Ng proteins and peptides eluted from antibody beads were applied on the MALDI plate. SIL‐Ng50‐78 was used as an internal standard for normalizing an intensity of each Ng peptide.ResultWe optimized the condition such as a kind of detergent and an amount of antibody beads in IP for enhancing Ng signal intensity in MS. In the examination of detergents, UDM showed the highest sensitivity among six tested detergents. Using IP‐MALDI‐MS, we tried to detect Ng peptides and proteins in human plasma. As a result, some peaks corresponding to a variety of Ng peptides (m/z m/z &gt; 5000.0) were detected in a plasma sample. To evaluate our semi‐quantitative assay using the internal standard, we selected some Ng peptides with low and high mass (m/z 2331.5–4084.7) to examine a linearity of the normalized intensity of Ng peptides. The accuracy in linear regression model showed within 80‐120% over a range of 5–160 pM in Ng48‐75 and 10‐160 pM in Ng50–78 and Ng33–75. Intra assay precision of Ng peptides in plasma was less than 20%CV.ConclusionIP‐MALDI‐MS enable the highly sensitive and simultaneous semi‐quantitative measurement of multiple Ng peptides levels. It could be helpful in the analysis of plasma Ng peptides for AD biomarker.

Detection of M-Protein in Acetonitrile Precipitates of Serum Using MALDI-TOF Mass Spectrometry: A Novel Methodology

Background: Multiple myeloma (MM) and plasmacytoma(s) belong to a group of clonal plasma cell dyscrasias. In 97-98% of all cases, they are characterised by the detection of a monoclonal protein (M-protein) in the blood, and sometimes in the urine. MALDI-TOF-mass spectrometry (MS) has demonstrated excellent analytical sensitivity for the screening and detection of M-protein. We present the results of a novel methodology for M-protein analysis by MALDI-TOF MS. Patients and Methods: Blood samples from patients and controls were collected after obtaining Institutional Ethics Committee approval. The work was carried out in accordance with the Declaration of Helsinki after obtaining written informed consent. Patients with confirmed multiple myeloma or plasmacytoma and M-protein detected by serum protein electrophoresis (SPEP), serum immunofixation electrophoresis (IFE), and serum free light chains (FLC) were included for MALDI-TOF MS analysis. IFE and FLC analysis were sent to independent laboratories for external validation of the MALDI-TOF MS results. Reagent-based extraction The serum fraction was separated from whole blood by centrifugation at 5000 rpm for 15 minutes and stored at -80oC until further analysis. Twenty-five μL of the serum sample was mixed with 50% acetonitrile (ACN) to form a precipitate. After precipitation and incubation, the mixture was centrifuged. The protein precipitate was washed with 20% ACN. After centrifugation, the supernatant was discarded, and the precipitate was reconstituted in a buffer comprising 10% formic acid (FA) and 50 mmol/L tris(2-carboxyethyl)phosphine hydrochloride (TCEP). The MALDI-TOF MS results were validated using immunoenrichment by anti-kappa (κ) and anti-lambda (λ) biotin-labelled antibodies immobilised on streptavidin magnetic beads. MALDI-TOF MS measurements were obtained for intact proteins using alpha-cyano-4-hydroxycinnamic acid as a matrix. The images obtained were overlaid on apparently healthy serum samples to confirm the presence of M-protein. The samples were then analysed using UltraflexTM LT, Bruker MALDI/TOF-TOF mass spectrometer. The mass spectra for each sample was exported to FlexAnalysis 3.3 (Bruker Daltonics) and background subtracted. A sample was considered positive for M-protein if there was a sharp or broad peak within the κ or λ mass/charge (m/z) range- κ m/z- [M+2H]2+: 11550-12300 Da; [M+H]+: 23100-24600 Da), and λ m/z- [M+2H]2+: 11100-11500 Da; [M+H]+: 22200-23100 Da. All the images were acquired at a m/z range of 10000-29000 Da. Mass measurement was analysed with a summation of 500-5000 shots depending on the intensity of the M-peak. Results: Twenty-seven patient samples: 24- multiple myeloma, and 3- plasmacytoma with an M-protein identified by other biochemical tests, were chosen for ACN precipitation and analysed by MALDI-TOF MS. The median age was 62 years (range:44-72); males-12 (44%). A mass spectrometrist, S.J was blinded to the IFE and FLC results- blinded analyst. N.M was the unblinded analyst. Neat sample (without dilution) was spotted on the MALDI plate for all the control and patient samples. The Gaussian distribution of κ and λ light chains were obtained by analysing 20 serum samples of apparently healthy blood donors. All the 27 samples (100%) with M-protein confirmed by the other biochemical techniques, demonstrated a peak suggestive of M-protein with mass/charge (m/z) falling within the κ or λ range on MALDI-TOF MS: 24 patients with κ peak, and 3 with λ peak. (Fig. 1a and 1b) Immunoenrichment was performed on two samples- 1 with κ peak, and the other with λ peak, analysed by MALDI-TOF-MS by ACN precipitation. The mass spectra by immunoenrichment and ACN precipitation were found to be identical with the light chain m/z falling within their respective range. (Fig. 2 and 3) Three samples were labelled as confounders due to low peak intensity. However, their peaks matched their corresponding IFE and FLC reports. Concordance between MALDI-TOF MS and IFE was observed in 21/23 patients (91%); concordance between MALDI-TOF MS and FLC was observed in 23/24 patients (96%). Conclusions: We report the results of a low-cost, reagent-based extraction process using ACN precipitation to enrich for κ and λ light chains, which can be used for screening and for qualitative analysis of M-protein. Further studies are required to identify the immunoglobulin isotype, and to quantify the M-protein by this methodology. Disclosures No relevant conflicts of interest to declare.

Automation and validation of a MALDI-TOF MS (Mass-Fix) replacement of immunofixation electrophoresis in the clinical lab.

A matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) method (Mass-Fix) as a replacement for gel-based immunofixation (IFE) has been recently described. To utilize Mass-Fix clinically, a validated automated method was required. Our aim was to automate the pre-analytical processing, improve positive specimen identification and ergonomics, reduce paper data storage and increase resource utilization without increasing turnaround time. Serum samples were batched and loaded onto a liquid handler along with reagents and a barcoded sample plate. The pre-analytical steps included: (1) Plating immunopurification beads. (2) Adding 10μl of serum. (3) Bead washing. (4) Eluting the immunoglobulins (Igs), and reducing to separate the heavy and light Ig chains. The resulting plate was transferred to a second low-volume liquid handler for MALDI plate spotting. MALDI-TOF mass spectra were collected. Integrated in-house developed software was utilized for sample tracking, driving data acquisition, data analysis, history tracking, and result reporting. A total of 1,029 residual serum samples were run using the automated system and results were compared to prior electrophoretic results. The automated Mass-Fix method was capable of meeting the validation requirements of concordance with IFE, limit of detection (LOD), sample stability and reproducibility with a low repeat rate. Automation and integrated software allowed a single user to process 320 samples in an 8h shift. Software display facilitated identification of monoclonal proteins. Additionally, the process maintains positive specimen identification, reduces manual pipetting, allows for paper free tracking, and does not significantly impact turnaround time (TAT). Mass-Fix is ready for implementation in a high-throughput clinical laboratory.

Practical guide on MALDI-TOF MS method development for high throughput profiling of pharmaceutically relevant, small molecule chemical reactions

High-throughput experimentation (HTE) offers means to allow medicinal chemists to synthesize a variety of pharmaceutically active compounds in an ultra-high throughput fashion, which also requires the development of an ultra-fast analytical method in order to reduce the bottleneck of post-reaction data analysis. We recently demonstrated MALDI-TOF-MS as an ultra-fast screening tool for the profiling of nano-mole C–N coupling reactions in a 1536 well plate at the rate of 0.3–0.4 s per sample. This work will cover MALDI method development steps including determination and optimization of sample concentration, spotting and drying methods, matrix selection, and quantitation approaches for high throughput pharmaceutical reaction profiling. The purpose of this work is to serve as a practical guide for organic and medicinal chemists to quickly develop a high throughput MALDI-TOF-MS method for routine screening of multi-well plate based chemical reactions without any MALDI plate modification or product tagging.

Comparison of MALDI-TOF Mass Spectrometry Analysis of Peripheral Blood and Bone Marrow Based Flow Cytometry for Tracking Measurable Residual Disease (MRD) in Patients with Multiple Myeloma

Comparison of MALDI-TOF Mass Spectrometry Analysis of Peripheral Blood and Bone Marrow Based Flow Cytometry for Tracking Measurable Residual Disease (MRD) in Patients with Multiple Myeloma

Mechanistic Studies on Cationization in MALDI-MS Employing a Split Sample Plate Set-up.

In the analysis of polymers by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), a commonly observed ionization pathway is cation-adduct formation, as polymers often lack easily ionizable (basic/acidic) functional groups. The mechanism of this process has been hypothesized to involve gas-phase cation attachment. In previous experiments, a split sample plate set-up has been introduced, enabling separate deposition of the components on individual MALDI plates. The plates are divided by a small gap of a few micrometers, allowing simultaneous laser irradiation from both plates, while precluding the possibility of any other interactions prior to ablation. Here, we extend on these studies by using different polymer-salt combinations to test the generalizability of a gas-phase ionization process. Clear evidence for in-plume ionization is presented for the model polymers poly (methyl methacrylate) and polystyrene. Furthermore, the contribution of in-plume processes to the overall ion formation by cationization is gauged, providing a first estimate for the importance of this pathway.

A pyrene linked peptide probe for quantitative analysis of protease activity via MALDI-TOF-MS

We report herein a rationally designed pyrene linked substrate for quantitative protease activity assay via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In this proof-of-concept study, a trypsin-specific peptide with the sequence of GGGGRG was selected to conjugate with pyrene forming a pyrene linked peptide probe, Py-GGGGRG. In the presence of trypsin, the Py-GGGGRG probe can be specifically hydrolyzed into Py-GGGGR. The introduction of pyrene greatly increased ionization efficiency of Py-peptides, and Py-peptides could be selectively captured from complex mixtures by a facially fabricated polystyrene coated MALDI plate through hydrophobic and π-π stacking interactions. As a result, trypsin activity can be directly quantified by relative intensity ratio of product and substrate via MALDI-TOF-MS without the use of external internal standard. A linear range of 0.1–10 μg/mL and a relatively low detection limit of 29 ng/mL were obtained. This method has also been successfully used for quantification of trypsin activity in urine and screening the inhibitors of trypsin. Besides, the proposed strategy was also validated for another protease, chymotrypsin, by using the probe Py-GGGGGGYG. Therefore, owing to simplicity, high-throughput capacity and quantificational accuracy, the proposed method shows great potential for activity assay of various proteases and screening their inhibitors via application of specific peptide sequences.

Case study: monitoring of Glc4 tetrasaccharide in the urine of Pompe patients, use of MALDI-TOF MS, and 1H NMR

Due to the lack of the acid 1,4-α-glucosidase, the urine of Pompe patients contains increased levels of specific glucose tetrasaccharide αGlc(1 → 6)αGlc(1 → 4)αGlc(1 → 4)Glc (Glc4), which was approved as a non-invasive biomarker of glycogen accumulation in the muscles of Pompe patients. MALDI-TOF mass spectrometry and 1H NMR spectroscopy were used for Glc4 detection in the urine samples of four Pompe patients with infantile, juvenile, and adult forms of the disease, before and during, or before and after, enzyme replacement therapy. The increased sensitivity and decreased urine volume required for Glc4 detection by MALDI-TOF mass spectrometry was obtained by specific derivatization directly on the MALDI plate by 3-aminoquinoline. This method is a suitable and fast method for Pompe screening. 1H NMR spectra, used for precise Glc4 quantifications, were proposed for the enzyme replacement therapy efficiency monitoring. Besides the clinical features and the biochemical parameters, knowledge about the evolution of the Glc4 levels over time during the administration of the therapy could help monitoring of the patient outcome as part of the standard protocol.

Magnetic carbon nanocomposites as a MALDI co-matrix enhancing MS-based glycomics.

More than 50% of all known proteins are glycosylated, which is critical for many biological processes such as protein folding and signal transduction. Glycosylation has proven to be associated with different mammalian diseases such as breast and liver cancers. Therefore, characterization of glycans is highly important to facilitate a better understanding of the development and progression of many human diseases. Although matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) offers several advantages such as ease of operation and short analysis times, however, due to the complexity of glycan structures and their low ionization efficiency, there are still challenges that need to be addressed to achieve sensitive glycan analysis. Here, magnetic carbon nanocomposites (CNPs@Fe3O4 NCs) were used as a new MALDI matrix or co-matrix for the analysis of glycans derived from different model glycoproteins and human blood serum samples. The addition of CNPs@Fe3O4 NCs to the matrix significantly enhanced glycan signal intensity by several orders of magnitude, and effectively controlled/reduced/eliminated in-source decay (ISD) fragmentation. The latter was attained by modulating CNPs@Fe3O4 NCs concentrations and allowed the simultaneous study of intact and fragmented glycans, and pseudo-MS3 analysis. Moreover, CNPs@Fe3O4 NCs was also effectively employed to desalt samples directly on MALDI plate, thus enabling direct MALDI-MS analysis of unpurified permethylated glycans derived from both model glycoproteins and biological samples. On-plate desalting enhanced sensitivity by reducing sample loss. Graphical abstract ᅟ.

Abstract LB-177: Quantification of cell signaling proteins by immuno-MALDI

Abstract Introduction. To improve cancer patient stratification and overcome the drawbacks of currently used approaches to assess signaling pathway proteins, we set out to develop immuno-matrix assisted laser ionization/desorption (iMALDI) assays combined with a phosphatase-based phosphopeptide quantitation (PPQ) approach to measure PI3K/AKT/mTOR pathway activity. Specifically, we targeted the C-terminal tryptic peptides of AKT1 and AKT2 due to their involvement in full kinase activation. Methods. Following trypsin digestion of cell lysate, stable isotope-labeled standard (SIS) peptides are added. After splitting the solution, one aliquot is treated with phosphatase. Bead-coupled antibodies enrich the non-phosphorylated target peptides, which are washed and spotted onto a MALDI plate, and acidic MALDI matrix elutes the peptides. The resulting light/heavy ratios of both aliquots allow the calculation of protein expression levels and peptide phosphorylation stoichiometry. Results. The iMALDI assays were validated for linear range and accuracy, as well as interference screening, requiring only ~50 µg of total cell or tissue lysate protein to quantify both AKT1 and AKT2 expression levels and phosphorylation stoichiometry. CVs of technical replicates were found to consistently be below 10%. We were able to quantify AKT1 and AKT2 from various cell lines and fresh frozen tumor samples, including SW480 and HCT116 colon cancer cell lines, MDA-MB-231 breast cancer cells, and colon cancer and breast cancer tumor lysates. Further, a direct comparison of matched fresh frozen and formalin-fixed paraffin-embedded (FFPE) tissues showed that the expression levels and phosphorylation stoichiometry quantified differ depending on the tissue preservation method, and that phosphorylation stoichiometries above 30% occur in only a small subset of samples. A direct comparison of four pairs of normal and adjacent tumor tissues showed elevated AKT1 phosphorylation stoichiometry of ~40% in a colorectal cancer liver metastasis, and significantly elevated AKT1 (3.6-fold) and AKT2 (2.2-fold) expression levels in a surgical breast tumor sample. Future directions. In a next step, patient-derived mouse xenograft tissue samples, collected after specific drug treatment, will be analyzed, and the AKT results will be correlated to genomic data to answer the hypothesis whether the AKT expression levels and phosphorylation stoichiometries correlate with response to treatment. Citation Format: Robert Popp, René P. Zahedi, André LeBlanc, Yassene Mohammed, Adriana Aguilar-Mahecha, Oliver Pötz, Mark Basik, Gerald Batist, Christoph H. Borchers. Quantification of cell signaling proteins by immuno-MALDI [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-177.