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The occurrence of a spontaneous nephropathy with intranuclear inclusions in laboratory mice has puzzled pathologists for over 4 decades, because its etiology remains elusive. The condition is more severe in immunodeficient animals, suggesting an infectious cause. Using metagenomics, we identify the causative agent as an atypical virus, termed "mouse kidney parvovirus" (MKPV), belonging to a divergent genus of Parvoviridae. MKPV was identified in animal facilities in Australia and North America, is transmitted via a fecal-oral or urinary-oral route, and is controlled by the adaptive immune system. Detailed analysis of the clinical course and histopathological features demonstrated a stepwise progression of pathology ranging from sporadic tubular inclusions to tubular degeneration and interstitial fibrosis and culminating in renal failure. In summary, we identify a widely distributed pathogen in laboratory mice and establish MKPV-induced nephropathy as a new tool for elucidating mechanisms of tubulointerstitial fibrosis that shares molecular features with chronic kidney disease in humans.
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Nefrite Intersticial/virologia , Parvovirus/isolamento & purificação , Parvovirus/patogenicidade , Animais , Austrália , Progressão da Doença , Feminino , Fibrose/patologia , Fibrose/virologia , Humanos , Rim/metabolismo , Rim/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nefrite Intersticial/fisiopatologia , América do Norte , Infecções por Parvoviridae/metabolismoRESUMO
Microsampling of patient blood promises several benefits over conventional phlebotomy practices to facilitate precision medicine studies. These include at-home patient blood collection, supporting telehealth monitoring, minimal postcollection processing, and compatibility with nonrefrigerated transport and storage. However, for proteomic biomarker studies, mass spectrometry of whole blood has generally been avoided in favor of using plasma or serum obtained from venepuncture. We evaluated the use of a volumetric absorptive microsampling (VAMS) device as a sample preparation matrix to enable LC-MS proteomic analyses of dried whole blood. We demonstrated the detection and robust quantitation of up to 1600 proteins from single-shot shotgun-LC-MS analysis of dried whole blood, greatly enhancing proteome depth compared with conventional single-shot LC-MS analyses of undepleted plasma. Some proteins not previously reported in blood were detected using this approach. Various washing reagents were used to demonstrate that proteins can be preferentially removed from VAMS devices prior to downstream analyses. We provide a demonstration that archival frozen blood cell pellets housed under long-term storage (exceeding 5 years) are compatible with VAMS to enable quantitation of potential biomarker proteins from biobank repositories. These demonstrations are important steps in establishing viable analysis workflows to underpin large-scale precision medicine studies. Data are available via ProteomeXchange with the identifier PXD028605.
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Proteômica , Espectrometria de Massas em Tandem , Biomarcadores , Coleta de Amostras Sanguíneas/métodos , Teste em Amostras de Sangue Seco/métodos , Humanos , Medicina de Precisão , Espectrometria de Massas em Tandem/métodosRESUMO
Mouse kidney parvovirus (MKPV) is a member of the provisional genus Chapparvovirus that causes renal disease in immune-compromised mice, with a disease course reminiscent of polyomavirus-associated nephropathy in immune-suppressed kidney transplant patients. Here we map four major MKPV transcripts, created by alternative splicing, to a common initiator region, and use mass spectrometry to identify "p10" and "p15" as novel chapparvovirus accessory proteins produced in MKPV-infected kidneys. p15 and the splicing-dependent putative accessory protein NS2 are conserved in all near-complete amniote chapparvovirus genomes currently available (from mammals, birds and a reptile). In contrast, p10 may be encoded only by viruses with >60% amino acid identity to MKPV. We show that MKPV is kidney-tropic and that the bat chapparvovirus DrPV-1 and a non-human primate chapparvovirus, CKPV, are also found in the kidneys of their hosts. We propose, therefore, that many mammal chapparvoviruses are likely to be nephrotropic.
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Rim/virologia , Infecções por Parvoviridae/veterinária , Infecções por Parvoviridae/virologia , Parvovirinae/fisiologia , Doenças dos Roedores/virologia , Proteínas Virais/metabolismo , Tropismo Viral , Animais , Humanos , Camundongos , Parvovirinae/genética , Proteínas Virais/genéticaRESUMO
RATIONALE: Matrix-assisted laser desorption ionisation with mass spectrometry imaging (MSI) has seen rapid development in recent years and as such is becoming an important technique for the mapping of biomolecules from the surface of tissues. One key area of development is the optimisation of analyte extraction by using modified matrices or mixes of common ones. METHODS: A series of serial sections were prepared for lipid MSI by either dry coating (sublimation) or by wet spray application of several matrices. These samples were then evaluated for analyte extraction, delocalisation and dynamic range. RESULTS: We have shown that the spraying and sublimation methods of matrix application can be used complementarily. This creates large datasets, with each preparation method applied narrowly and then interpreted as a 'fraction' of the whole. Once combined, the dynamic range is significantly increased. We have dubbed this technique 'matrix phase fractionation'. CONCLUSIONS: We have found that, by utilising matrix phase fractionation for the detection of lipids in brain tissue, it is possible to create a significantly more comprehensive dataset than would otherwise be possible with traditional 'single-run' workflows.
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Fibroblast activation protein-alpha (FAP) is a cell-surface transmembrane-anchored dimeric protease. This unique, constitutively active serine protease has both dipeptidyl aminopeptidase and endopeptidase activities and can hydrolyze the post-proline bond. FAP expression is very low in adult organs but is upregulated by activated fibroblasts in sites of tissue remodeling, including fibrosis, atherosclerosis, arthritis and tumors. To identify the endogenous substrates of FAP, we immortalized primary mouse embryonic fibroblasts (MEFs) from FAP gene knockout embryos and then stably transduced them to express either enzymatically active or inactive FAP. The MEF secretomes were then analyzed using degradomic and proteomic techniques. Terminal amine isotopic labeling of substrates (TAILS)-based degradomics identified cleavage sites in collagens, many other extracellular matrix (ECM) and associated proteins, and lysyl oxidase-like-1, CXCL-5, CSF-1, and C1qT6, that were confirmed in vitro In addition, differential metabolic labeling coupled with quantitative proteomic analysis also implicated FAP in ECM-cell interactions, as well as with coagulation, metabolism and wound healing associated proteins. Plasma from FAP-deficient mice exhibited slower than wild-type clotting times. This study provides a significant expansion of the substrate repertoire of FAP and provides insight into the physiological and potential pathological roles of this enigmatic protease.
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Fibroblastos/citologia , Gelatinases/genética , Gelatinases/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteômica/métodos , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Adipocinas/sangue , Adipocinas/química , Aminoácido Oxirredutases/sangue , Aminoácido Oxirredutases/química , Animais , Técnicas de Cultura de Células , Linhagem Celular , Quimiocina CXCL5/sangue , Quimiocina CXCL5/química , Endopeptidases , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Humanos , Fator Estimulador de Colônias de Macrófagos/sangue , Fator Estimulador de Colônias de Macrófagos/química , Camundongos , Mapas de Interação de Proteínas , Proteólise , Especificidade por SubstratoRESUMO
The differentiation of human adipose derived stem cells toward a neural phenotype by small molecules has been a vogue topic in the last decade. The characterization of the produced cells has been explored on a broad scale, examining morphological and specific surface protein markers; however, the lack of insight into the expression of functional proteins and their interactive partners is required to further understand the extent of the process. The phenotypic characterization by proteomic profiling allows for a substantial in-depth analysis of the molecular machinery induced and directing the cellular changes through the process. Herein we describe the temporal analysis and quantitative profiling of neural differentiating human adipose-derived stem cells after sub-proteome enrichment using a bisindolylmaleimide chemical probe. The results show that proteins enriched by the Bis-probe were identified reproducibly with 133, 118, 126 and 89 proteins identified at timepoints 0, 1, 6 and 12, respectively. Each temporal timepoint presented several shared and unique proteins relative to neural differentiation and their interactivity. The major protein classes enriched and quantified were enzymes, structural and ribosomal proteins that are integral to differentiation pathways. There were 42 uniquely identified enzymes identified in the cells, many acting as hubs in the networks with several interactions across the network modulating key biological pathways. From the cohort, it was found by gene ontology analysis that 18 enzymes had direct involvement with neurogenic differentiation.
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Biomarcadores/metabolismo , Diferenciação Celular , Indóis/metabolismo , Maleimidas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Neurogênese , Proteoma/metabolismo , Inibidores Enzimáticos/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Fenótipo , Proteoma/análise , Proteoma/químicaRESUMO
Reproducibility has been one of the biggest hurdles faced when attempting to develop quantitative protocols for MALDI mass spectrometry. The heterogeneous nature of sample recrystallization has made automated sample acquisition somewhat "hit and miss" with manual intervention needed to ensure that all sample spots have been analyzed. In this review, we explore the last 30 years of literature and anecdotal evidence that has attempted to address and improve reproducibility in MALDI MS. Though many methods have been attempted, we have discovered a significant publication history surrounding the use of nitrocellulose as a substrate to improve homogeneity of crystal formation and therefore reproducibility. We therefore propose that this is the most promising avenue of research for developing a comprehensive and universal preparation protocol for quantitative MALDI MS analysis. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 37:217-228, 2018.
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RATIONALE: This paper, in conjunction with a work published earlier this year by O'Rourke et al., aims to provide a comprehensive set of protocols for the analysis of formalin-fixed paraffin-embedded (FFPE) tissue via matrix-assisted laser desorption/ionisation imaging mass spectrometry (MALDI IMS) in a low-cost and highly repeatable and robust way, thereby allowing other research teams to begin their own IMS-centered avenues of research. METHODS: Samples of FFPE tissue were sectioned at 5 µm, water float mounted to specially prepared ITO glass slides and then dilipidated in a graded alcohol series. Tissue sections were then antigen retrieved under pressure in 20 mmol Tris-HCl (pH 8.8), coated with trypsin and digested O/N at 37°C. Samples were then sublimated with matrix to a final coverage of 0.2 mg/cm(2), recrystallised at 37°C with 50:50 acetonitrile (ACN)/0.1% trifluoroacetic acid (TFA) for 1 h and analysed with a MALDI TOF/TOF mass spectrometer. RESULTS: Serial sections were imaged, revealing little to no variation with regards to image quality and corresponding spectra. We have also attempted to describe the processes that govern the various aspects of this protocol with respect to each step necessary to ensure reproducibility. CONCLUSIONS: We are confident that this protocol in conjunction with the work published earlier by O'Rourke et al. provides the basis for a repeatable and robust protocol for the analysis of tissues from various sources via MALDI-IMS. The descriptions of key steps within allows for easy adoption of the protocol while allowing for desired modifications to be performed with minimal yet intuitive adjustment.
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Histocitoquímica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Medula Espinal/química , Formaldeído , Humanos , Inclusão em Parafina , TripsinaRESUMO
RATIONALE: There are currently multiple methods available for the preparation of fresh frozen tissue samples for analysis via matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) imaging mass spectrometry (IMS). Although these methods report excellent results, many are expensive automated approaches. With no published attempt to standardise less expensive manual processes, our work aims to provide a robust and repeatable method of sample preparation for MALDI-TOF-IMS that is applicable to a variety of tissue types, well explained, simple and cost effective. METHODS: Fresh frozen tissue was sectioned at 12 µm and mounted onto liquid nitrocellulose coated slides, washed in a graded alcohol series and then mounted into a modified sublimation apparatus. Matrix is deposited onto the slide to achieve a desired coating of 0.2 mg/cm(2). Once coated, the slide is mounted into a custom-built vapor chamber and recrystallised with 50% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA) for 1 h at 37°C. The slide is then analysed using MALDI-IMS. RESULTS: We have successfully implemented this method for a host of tissue samples, including brain, liver, kidney and heart, with no variation in relative spectra or processing method required. When the protocol is followed correctly, sublimations and recrystallisations are highly predictable with limited variation between samples and a very low failure rate. Additional apparatuses can be easily constructed by following the included instructions, that perform as per specifications with no variation. CONCLUSIONS: We believe that we have described a complete protocol for MALDI-IMS that is easy to use and highly reproducible. The lack of expensive commercially available equipment makes this process very cheap with a relatively low initial outlay and our hope is that more laboratories will begin IMS-based avenues of research based on the work we have performed.
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Secções Congeladas/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Química Encefálica , Cristalização , Secções Congeladas/economia , Modelos Teóricos , RatosRESUMO
Biofluids such as blood plasma are rich reservoirs of potential biomarkers for disease diagnosis, prognosis, and prediction of treatment response. However, mass spectrometry analysis of circulating plasma proteins remains challenging. The introduction of data-independent acquisition mass spectrometry (DIA-MS) is an important step toward addressing detection of less abundant plasma proteins. Numerous plasma peptide MS/MS spectral library datasets produced from extensive plasma fractionation are accessible from public archives, and these can be repurposed as spectral reference libraries to increase the depth of proteomic analysis when DIA-MS is used. Here we describe the workflow that relies on reusing the existing spectral reference libraries by populating them with locally obtained peptide MS/MS data acquired by DIA-MS. This approach was demonstrated effectively to identify putative plasma biomarkers of response to neoadjuvant chemotherapy in the setting of pancreatic ductal adenocarcinoma (PDAC) (O'Rourke et al., J Proteomics 231:103998, 2021).
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Biomarcadores Tumorais , Neoplasias Pancreáticas , Proteômica , Espectrometria de Massas em Tandem , Humanos , Biomarcadores Tumorais/sangue , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/sangue , Biblioteca de Peptídeos , Carcinoma Ductal Pancreático/sangue , Carcinoma Ductal Pancreático/tratamento farmacológico , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/metabolismo , Terapia Neoadjuvante/métodosRESUMO
Chronic obstructive pulmonary disease (COPD) ranks as the third leading cause of global illness and mortality. It is commonly triggered by exposure to respiratory irritants like cigarette smoke or biofuel pollutants. This multifaceted condition manifests through an array of symptoms and lung irregularities, characterized by chronic inflammation and reduced lung function. Present therapies primarily rely on maintenance medications to alleviate symptoms, but fall short in impeding disease advancement. COPD's diverse nature, influenced by various phenotypes, complicates diagnosis, necessitating precise molecular characterization. Omics-driven methodologies, including biomarker identification and therapeutic target exploration, offer a promising avenue for addressing COPD's complexity. This analysis underscores the critical necessity of improving molecular profiling to deepen our comprehension of COPD and identify potential therapeutic targets. Moreover, it advocates for tailoring treatment strategies to individual phenotypes. Through comprehensive exploration-based molecular characterization and the adoption of personalized methodologies, innovative treatments may emerge that are capable of altering the trajectory of COPD, instilling optimism for efficacious disease-modifying interventions.
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PURPOSE: Robust, affordable plasma proteomic biomarker workflows are needed for large-scale clinical studies. We evaluated aspects of sample preparation to allow liquid chromatography-mass spectrometry (LC-MS) analysis of more than 1500 samples from the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) trial of adults with type 2 diabetes. METHODS: Using LC-MS with data-independent acquisition we evaluated four variables: plasma protein depletion, EDTA or citrated anti-coagulant blood collection tubes, plasma lipid depletion strategies and plasma freeze-thaw cycles. Optimised methods were applied in a pilot study of FIELD participants. RESULTS: LC-MS of undepleted plasma conducted over a 45 min gradient yielded 172 proteins after excluding immunoglobulin isoforms. Cibachrome-blue-based depletion yielded additional proteins but with cost and time expenses, while immunodepleting albumin and IgG provided few additional identifications. Only minor variations were associated with blood collection tube type, delipidation methods and freeze-thaw cycles. From 65 batches involving over 1500 injections, the median intra-batch quantitative differences in the top 100 proteins of the plasma external standard were less than 2%. Fenofibrate altered seven plasma proteins. CONCLUSIONS AND CLINICAL RELEVANCE: A robust plasma handling and LC-MS proteomics workflow for abundant plasma proteins has been developed for large-scale biomarker studies that balance proteomic depth with time and resource costs.
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Diabetes Mellitus Tipo 2 , Fenofibrato , Adulto , Humanos , Cromatografia Líquida/métodos , Fenofibrato/farmacologia , Fenofibrato/uso terapêutico , Proteômica/métodos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Projetos Piloto , Espectrometria de Massas em Tandem , Proteínas Sanguíneas/metabolismo , BiomarcadoresRESUMO
(1) Background: MALDI imaging is a technique that still largely depends on time of flight (TOF)-based instrument such as the Bruker UltrafleXtreme. While capable of performing targeted MS/MS, these instruments are unable to perform fragmentation while imaging a tissue section necessitating the reliance of MS1 values for peptide level identifications. With this premise in mind, we have developed a hybrid bioinformatic/image-based method for the identification and validation of viral biomarkers. (2) Methods: Formalin-Fixed Paraffin-Embedded (FFPE) mouse samples were sectioned, mounted and prepared for mass spectrometry imaging using our well-established methods. Peptide identification was achieved by first extracting confident images corresponding to theoretical viral peptides. Next, those masses were used to perform a Peptide Mmass Fingerprint (PMF) searched against known viral FASTA sequences against a background mouse FASTA database. Finally, a correlational analysis was performed with imaging data to confirm pixel-by-pixel colocalization and intensity of viral peptides. (3) Results: 14 viral peptides were successfully identified with significant PMF Scores and a correlational result of >0.79 confirming the presence of the virus and distinguishing it from the background mouse proteins. (4) Conclusions: this novel approach leverages the power of mass spectrometry imaging and provides confident identifications for viral proteins without requiring MS/MS using simple MALDI Time Of Flight/Time Of Flight (TOF/TOF) instrumentation.
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Pathogenic mycobacteria inhibit inflammasome activation to establish infection. Although it is known that potassium efflux is a trigger for inflammasome activation, the interaction between mycobacterial infection, potassium efflux, and inflammasome activation has not been investigated. Here, we use Mycobacterium marinum infection of zebrafish embryos and Mycobacterium tuberculosis infection of THP-1 cells to demonstrate that pathogenic mycobacteria up-regulate the host WNK signalling pathway kinases SPAK and OXSR1 which control intracellular potassium balance. We show that genetic depletion or inhibition of OXSR1 decreases bacterial burden and intracellular potassium levels. The protective effects of OXSR1 depletion are at least partially mediated by NLRP3 inflammasome activation, caspase-mediated release of IL-1ß, and downstream activation of protective TNF-α. The elucidation of this druggable pathway to potentiate inflammasome activation provides a new avenue for the development of host-directed therapies against intracellular infections.
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Inflamassomos , Mycobacterium , Animais , Inflamassomos/metabolismo , Mycobacterium/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Potássio/metabolismo , Transdução de Sinais , Peixe-ZebraRESUMO
The detection of disease-related plasma biomarkers has challenged the proteomic community for years. Attractive features for plasma proteomics includes the ease of collection and small volume needed for analysis, but on the other hand, the presence of highly abundant proteins complicates sample preparation procedures and reduces dynamic range. Data independent acquisition label free quantitation (DIA-LFQ) by mass spectrometry partly overcomes the dynamic range issue; however, generating the peptide spectral reference libraries that allow extensive analysis of the plasma proteome can be a slow and expensive task which is unattainable for many laboratories. We investigated the re-purposing of publically available plasma proteome datasets and the impact on peptide/protein detection for DIA-LFQ. We carried out these studies in the context of identifying putative biomarkers of response to neoadjuvant chemotherapy (NAC) for pancreatic ductal adenocarcinoma, as no useful plasma biomarkers have been clinically adopted. We demonstrated the benefit in searching DIA data against multiple spectral libraries to show that complement proteins were linked to NAC response in PDAC patients, confirming previous observations of the prognostic utility of complement following adjuvant chemotherapy. Our workflow demonstrates that DIA-LFQ can be readily applied in the oncology setting for the putative assignment of clinically relevant plasma biomarkers. STATEMENT OF SIGNIFICANCE: The proteomic mass spectrometry analysis of undepleted, unfractionated human plasma has benefits for sample throughput but remains challenging to obtain deep coverage. This work evaluated the re-purposing of open source peptide mass spectrometry data from human plasma to create spectral reference libraries for use in Data independent acquisition (DIA). We showed how seeding in locally acquired data to integrate iRT peptides into spectral libraries increased identification confidence by facilitating querying of multiple libraries. This workflow was applied to the discovery of putative plasma biomarkers for response to neoadjuvant chemotherapy (NAC) in pancreatic ductal adenocarcinoma patients. There is a paucity of prior information in the literature on this topic and we show that good responder patients have reduced levels of complement proteins.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Biomarcadores , Carcinoma Ductal Pancreático/tratamento farmacológico , Humanos , Terapia Neoadjuvante , Neoplasias Pancreáticas/tratamento farmacológico , ProteômicaRESUMO
Peer review is the way in which we, as scientists, criticise, check, and confirm the findings of our colleagues. The process of peer review relies on individuals in all fields applying their particular expertise and determining if they agree with the findings submitted for publication. In recent years, there has been a significant rise in the number of manuscripts submitted for publication that draw from a range of disparate and complementary fields. This has created the curious situation where an expert may be requested to review a manuscript that is only partially within their immediate field of expertise. The issue that arises is that, without full knowledge of the data, techniques, methodologies, and principles that are presented, it is difficult for reviewers to make properly informed decisions, especially when it can take an entire career to reach that specific level of expertise in a single field. From this perspective, we explore these issues and also provide a commentary on how peer review could evolve in the context of a changing cross-disciplinarily-focused scientific landscape.
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The array of analytes that can be measured by MADLI MS has created an equally vast range of calibration mixtures. The inherent problem with this is that acquiring all of them at commercial rates can be prohibitively expensive. With this in mind, we have created a low-cost alternative to the most commonly used peptide calibrants. We were able to achieve an overall 78 ppm mass accuracy across a mass range of 900 to 2500 Da which was comparable to the mass accuracy achievable with commercial peptide mixes and hence has become a viable alternative.
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Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is a well-established technique for elucidating the location and relative abundance of a range of biomolecules. More recently, research into this technique has shifted from simple discovery and demonstration of utility to application in biomedical research. Here, we describe a protocol utilizing MALDI-IMS for the spatial mapping of lipids in brain tissue from normal human brains and brains from patients with Alzheimer's disease, in the context of Alzheimer's disease. Improved accuracy calibration of the instrument from the tissue surface is emphasized, as this allows for significantly improved mass determination in time of flight (TOF)-based instruments enabling more confident preliminary lipid identification. This improved initial result allows MALDI-IMS data to be complemented with additional instrumentation, such as liquid chromatography mass spectrometry workflows or specialized non-TOF systems such as Fourier transform cyclotron resonance instruments. This method is not limited to human tissue and can be applied to virtually any lipid-rich formalin-fixed tissue. © 2019 by John Wiley & Sons, Inc.
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Doença de Alzheimer/patologia , Química Encefálica , Encéfalo/patologia , Lipídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , HumanosRESUMO
The accurate quantification of changes in the abundance of proteins is one of the main applications of proteomics. The maintenance of accuracy can be affected by bias and error that can occur at many points in the experimental process, and normalization strategies are crucial to attempt to overcome this bias and return the sample to its regular biological condition, or normal state. Much work has been published on performing normalization on data post-acquisition with many algorithms and statistical processes available. However, there are many other sources of bias that can occur during experimental design and sample handling that are currently unaddressed. This article aims to cast light on the potential sources of bias and where normalization could be applied to return the sample to its normal state. Throughout we suggest solutions where possible but, in some cases, solutions are not available. Thus, we see this article as a starting point for discussion of the definition of and the issues surrounding the concept of normalization as it applies to the proteomic analysis of biological samples. Specifically, we discuss a wide range of different normalization techniques that can occur at each stage of the sample preparation and analysis process.
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The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.