Liquid MALDI MS analysis of complex peptide and proteome samples

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) is well-known to be a powerful technique for the analysis of biological samples. By using glycerol-based liquid support matrices (LSMs) instead of conventional MALDI matrices the power of this technique can be extended further. In this study, we exploited LSMs for the identification of complex samples, i.e. the Lactobacillus proteome and a bovine serum albumin (BSA) digest. Liquid and solid MALDI samples were manually and robotically prepared by coupling a nanoflow high performance liquid chromatography (nanoHPLC) system to an automated MALDI sample spotting device. MS and MS/MS data were successfully acquired at the femtomole level using TOF/TOF as well as Q-TOF instrumentation and used for protein identification searching sequence databases. For the BSA digest analysis, liquid MALDI samples resulted in peptide mass fingerprints which led to a higher confidence in protein identification compared to solid (crystalline) MALDI samples. However, post-source decay (PSD) MS/MS analysis of both the proteome of Lactobacillus plantarum WCFS1 cells and BSA digest showed that further optimization of the formation and detection of peptide fragment ions is still needed for liquid MALDI samples as the MS/MS ion search score was lower than for the solid MALDI samples, reflecting the poorer quality of the liquid MALDI-PSD spectra, which can be attributed to the differences in PSD parameters and their optimization that is currently achievable

Sample preparation: a crucial factor for the analytical performance of rationally designed MALDI matrices

Evidence is presented that the performance of the rationally designed MALDI matrix 4-chloro-α-cyanocinnamic acid (ClCCA) in comparison to its well-established predecessor α-cyano-4-hydroxycinnamic acid (CHCA) is significantly dependent on the sample preparation, such as the choice of the target plate. In this context, it becomes clear that any rational designs of MALDI matrices and their successful employment have to consider a larger set of physicochemical parameters, including sample crystallization and morphology/topology, in addition to parameters of basic (solution and/or gas-phase) chemistry

CCQM-K55.b (Aldrin) : Final report: october 2012. CCQM-K55.b key comparison on the characterization of organic substances for chemical purity

Under the auspices of the Organic Analysis Working Group (OAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) a key comparison, CCQM K55.b, was coordinated by the Bureau International des Poids et Mesures (BIPM) in 2010/2011. Nineteen national measurement institutes and the BIPM participated. Participants were required to assign the mass fraction of aldrin present as the main component in the comparison sample for CCQM-K55.b which consisted of technical grade aldrin obtained from the National Measurement Institute Australia that had been subject to serial recrystallization and drying prior to sub-division into the units supplied for the comparison. Aldrin was selected to be representative of the performance of a laboratory's measurement capability for the purity assignment of organic compounds of medium structural complexity [molar mass range 300 Da to 500 Da] and low polarity (pKOW < −2) for which related structure impurities can be quantified by capillary gas phase chromatography (GC). The key comparison reference value (KCRV) for the aldrin content of the material was 950.8 mg/g with a combined standard uncertainty of 0.85 mg/g. The KCRV was assigned by combination of KCRVs assigned by consensus from participant results for each orthogonal impurity class. The relative expanded uncertainties reported by laboratories having results consistent with the KCRV ranged from 0.3% to 0.6% using a mass balance approach and 0.5% to 1% using a qNMR method. The major analytical challenge posed by the material proved to be the detection and quantification of a significant amount of oligomeric organic material within the sample and most participants relying on a mass balance approach displayed a positive bias relative to the KCRV (overestimation of aldrin content) in excess of 10 mg/g due to not having adequate procedures in place to detect and quantify the non-volatile content—specifically the non-volatile organics content—of the comparison sample. There was in general excellent agreement between participants in the identification and the quantification of the total and individual related structure impurities, water content and the residual solvent content of the sample. The comparison demonstrated the utility of 1H NMR as an independent method for quantitative analysis of high purity compounds. In discussion of the participant results it was noted that while several had access to qNMR estimates for the aldrin content that were inconsistent with their mass balance determination they decided to accept the mass balance result and assumed a hidden bias in their NMR data. By contrast, laboratories that placed greater confidence in their qNMR result were able to resolve the discrepancy through additional studies that provided evidence of the presence of non-volatile organic impurity at the requisite level to bring their mass balance and qNMR estimates into agreement.Fil: Westwood, Steven. Bureau International des Poids et Mesures (BIPM); FranciaFil: Josephs, Ralf. Bureau International des Poids et Mesures (BIPM); FranciaFil: Choteau, Tiphaine. Bureau International des Poids et Mesures (BIPM); FranciaFil: Daireaux, Adeline. Bureau International des Poids et Mesures (BIPM); FranciaFil: Mesquida, Charline. Bureau International des Poids et Mesures (BIPM); FranciaFil: Wielgosz, Robert. Bureau International des Poids et Mesures (BIPM); FranciaFil: Rosso, Adriana. Instituto Nacional de Tecnología Industrial (INTI); ArgentinaFil: Ruiz de Arechavaleta, Mariana. Instituto Nacional de Tecnología Industrial (INTI); ArgentinaFil: Davies, Stephen. National Measurement Institute (NMIA); AustraliaFil: Wang, Hongjie. National Measurement Institute (NMIA); AustraliaFil: Pires do Rego, Eliane Cristina. Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMetro); BrasilFil: Marques Rodrigues, Janaína. Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMetro); BrasilFil: de Freitas Guimarães, Evelyn. Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMetro); BrasilFil: Barreto Sousa, Marcus Vinicius. Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMetro); BrasilFil: Monteiro, Tânia Maria. Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMetro); BrasilFil: Alves das Neves Valente, Laura. Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMetro); BrasilFil: Marques Violante, Fernando Gustavo. Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMetro); BrasilFil: Rubim, Renato. Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMetro); BrasilFil: Almeida, Ribeiro. Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMetro); BrasilFil: Baptista Quaresma, Maria Cristina. Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMetro); BrasilFil: Nogueira, Raquel. Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMetro); BrasilFil: Windust, Anthony. Institute for National Measurement Standards. National Research Council Canada (NRC-INMS); CanadáFil: Dai, Xinhua. National Institute of Metrology (NIM); ChinaFil: Li, Xiaomin. National Institute of Metrology (NIM); ChinaFil: Zhang, Wei. National Institute of Metrology (NIM); ChinaFil: Li, Ming. National Institute of Metrology (NIM); ChinaFil: Shao, Mingwu. National Institute of Metrology (NIM); ChinaFil: Wei, Chao. National Institute of Metrology (NIM); ChinaFil: Wong, Siu-kay. Government Laboratory of Hong Kong SAR (GLHK); ChinaFil: Cabillic, Julie. Laboratoire National de Métrologie et d’Essais (LNE); FranciaFil: Gantois, Fanny. Laboratoire National de Métrologie et d’Essais (LNE); FranciaFil: Philipp, Rosemarie. Bundesanstalt für Materialforschung (BAM); AlemaniaFil: Pfeifer, Dietmar. Bundesanstalt für Materialforschung (BAM); AlemaniaFil: Hein, Sebastian. Bundesanstalt für Materialforschung (BAM); AlemaniaFil: Klyk-Seitz, Urszula-Anna. Bundesanstalt für Materialforschung (BAM); AlemaniaFil: Ishikawa, Keiichiro. National Metrology Institute of Japan (NMIJ); JapónFil: Castro, Esther. Centro Nacional de Metrología (CENAM); MéxicoFil: Gonzalez, Norma. Centro Nacional de Metrología (CENAM); MéxicoFil: Krylov, Anatoly. D. I. Mendeleev Institute for Metrology (VNIIM); RusiaFil: Lin, Teo Tang. Health Sciences Authority (HSA); SingapurFil: Kooi, Lee Tong. Health Sciences Authority (HSA); SingapurFil: Fernandes-Whaley, M. National Metrology Institute of South Africa (NMISA); SudáfricaFil: Prévoo, D. National Metrology Institute of South Africa (NMISA); SudáfricaFil: Archer, M. National Metrology Institute of South Africa (NMISA); SudáfricaFil: Visser, R. National Metrology Institute of South Africa (NMISA); SudáfricaFil: Nlhapo, N. National Metrology Institute of South Africa (NMISA); SudáfricaFil: de Vos, B. National Metrology Institute of South Africa (NMISA); SudáfricaFil: Ahn, Seonghee. Korea Research Institute of Standards and Science (KRISS); Corea del SurFil: Pookrod, Preeyaporn. National Institute of Metrology of Thailand (NIMT); TailandiaFil: Wiangnon, Kanjana. National Institute of Metrology of Thailand (NIMT); TailandiaFil: Sudsiri, Nittaya. National Institute of Metrology of Thailand (NIMT); TailandiaFil: Muaksang, Kittiya. National Institute of Metrology of Thailand (NIMT); TailandiaFil: Cherdchu, Chainarong. National Institute of Metrology of Thailand (NIMT); TailandiaFil: Gören, Ahmet Ceyhan. National Metrology Institute (TUBITAK UME); TurquíaFil: Bilsel, Mine. National Metrology Institute (TUBITAK UME); TurquíaFil: LeGoff, Thierry. LGC Limited; Reino UnidoFil: Bearden, Dan. National Institute of Standards and Technology (NIST); Estados UnidosFil: Bedner, Mary. National Institute of Standards and Technology (NIST); Estados UnidosFil: Duewer, David. National Institute of Standards and Technology (NIST); Estados UnidosFil: Hancock, Diane. National Institute of Standards and Technology (NIST); Estados UnidosFil: Lang, Brian. National Institute of Standards and Technology (NIST); Estados UnidosFil: Lippa, Katrice. National Institute of Standards and Technology (NIST); Estados UnidosFil: Schantz, Michele. National Institute of Standards and Technology (NIST); Estados UnidosFil: Sieber, John. National Institute of Standards and Technology (NIST); Estados Unido

Coupling liquid MALDI MS to liquid chromatography

Matrix-assisted laser desorption/ionisation (MALDI) coupled with time-of-flight (TOF) mass spectrometry (MS) is a powerful tool for the analysis of biological samples, and nanoflow high-performance liquid chromatography (nanoHPLC) is a useful separation technique for the analysis of complex proteomics samples. The off-line combination of MALDI and nanoHPLC has been extensively investigated and straightforward techniques have been developed, focussing particularly on automated MALDI sample preparation that yields sensitive and reproducible spectra. Normally conventional solid MALDI matrices such as α-cyano-4-hydroxycinnamic acid (CHCA) are used for sample preparation. However, they have limited usefulness in quantitative measurements and automated data acquisition because of the formation of heterogeneous crystals, resulting in highly variable ion yields and desorption/ ionization characteristics. Glycerol-based liquid support matrices (LSM) have been proposed as an alternative to the traditional solid matrices as they provide increased shot-to-shot reproducibility, leading to prolonged and stable ion signals and therefore better results. This chapter focuses on the integration of the liquid LSM MALDI matrices into the LC-MALDI MS/MS approach in identifying complex and large proteomes. The interface between LC and MALDI consists of a robotic spotter, which fractionates the eluent from the LC column into nanoliter volumes, and co-spots simultaneously the liquid matrix with the eluent fractions onto a MALDI target plate via sheath flow. The efficiency of this method is demonstrated through the analysis of trypsin digests of both bovine serum albumin (BSA) and Lactobacillus plantarum WCFS1 proteins