Protocols

Non-Targeted Identification of D-Amino Acid-Containing Peptides through Enzymatic Screening, Chiral Amino Acid Analysis, and LC-MS
S. Okyem, E.V. Romanova, H.C. Tai, J.W. Checco, J.V. Sweedler, Methods Mol. Biol. 2758, 2024, 227–240.

Abstract: D-amino acid-containing peptides (DAACPs) in animals are a class of bioactive molecules formed via the posttranslational modification of peptides consisting of all-L-amino acid residues. Amino acid residue isomerization greatly impacts the function of the resulting DAACP. However, because isomerization does not change the peptide’s mass, this modification is difficult to detect by most mass spectrometry-based peptidomic approaches. Here we describe a method for the identification of DAACPs that can be used to systematically survey peptides extracted from a tissue sample in a nontargeted manner.

Bioinformatics for Prohormone and Neuropeptide Discovery
B.R. Southey, E.V. Romanova, S.L. Rodriguez-Zas, J.V. Sweedler, Methods Mol. Biol. 2758, 2024, 151–178.

Abstract: Neuropeptides and peptide hormones are signaling molecules produced via complex posttranslational modifications of precursor proteins known as prohormones. Neuropeptides activate specific receptors and are associated with the regulation of physiological systems and behaviors. The identification of prohormones—and the neuropeptides created by these prohormones—from genomic assemblies has become essential to support the annotation and use of the rapidly growing number of sequenced genomes. Here we describe a well-validated methodology for identifying the prohormone complement from genomic assemblies that employs widely available public toolsets and databases. The uncovered prohormone sequences can then be screened for putative neuropeptides to enable accurate proteomic discovery and validation.

Multiscale Biochemical Mapping of the Brain through Deep-Learning-Enhanced High-Throughput Mass Spectrometry
Y.R. Xie, D.C. Castro, S.S. Rubakhin, T.J. Trinklein, J.V. Sweedler, F. Lam, Nat. Methods 21, 2024, 521–530.

Abstract: Multimodal mass spectrometry (MMS) incorporates an imaging modality with probe-based mass spectrometry (MS) to enable precise, targeted data acquisition and provide additional biological and chemical data not available by MS alone. Two categories of MMS are covered; in the first, an imaging modality guides the MS probe to target individual cells and to reduce acquisition time by automatically defining regions of interest. In the second category, imaging and MS data are coupled in the data analysis pipeline to increase the effective spatial resolution using a higher resolution imaging method, correct for tissue deformation, and incorporate fine morphological features in an MS imaging dataset. Recent methodological and computational developments are covered along with their application to single-cell and imaging analyses.

Workflow for High-throughput Screening of Enzyme Mutant Libraries Using Matrix-assisted Laser Desorption/Ionization Mass Spectrometry Analysis of Escherichia coli Colonies
K. Choe and J.V. Sweedler, Bio-protocol 13, 2023, e4862.

Abstract: High-throughput molecular screening of microbial colonies and DNA libraries are critical procedures that enable applications such as directed evolution, functional genomics, microbial identification, and creation of engineered microbial strains to produce high-value molecules. A promising chemical screening approach is the measurement of products directly from microbial colonies via optically guided matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Measuring the compounds from microbial colonies bypasses liquid culture with a screen that takes approximately 5 s per sample. We describe a protocol combining a dedicated informatics pipeline and sample preparation method that can prepare up to 3,000 colonies in under 3 h. The screening protocol starts from colonies grown on Petri dishes and then transferred onto MALDI plates via imprinting. The target plate with the colonies is imaged by a flatbed scanner and the colonies are located via custom software. The target plate is coated with MALDI matrix, MALDI-MS analyzes the colony locations, and data analysis enables the determination of colonies with the desired biochemical properties. This workflow screens thousands of colonies per day without requiring additional automation. The wide chemical coverage and the high sensitivity of MALDI-MS enable diverse screening projects such as modifying enzymes and functional genomics surveys of gene activation/inhibition libraries.

High-throughput Image-guided Microprobe Mass Spectrometric Analysis of Single Cells
S.S. Rubakhin, E.V. Romanova, J.V. Sweedler, in: Single Cell ‘Omics of Neuronal Cells, edited by J.V. Sweedler, J.H. Eberwine, S.E. Fraser, Neuromethods, vol 184, Humana, New York, NY. 2022, 115–163.

Abstract: Characterizing the chemicals within individual cells is an important analytical capability, with mass spectrometry (MS) being one of the most chemically information-rich techniques available. There are two major approaches for characterizing single cells using MS: the assay of small-volume solutions using electrospray ionization and, the focus of this chapter, measurement of cells dispersed across a surface using matrix-assisted laser desorption/ionization (MALDI) MS. In combination with spatiometric information obtained via optical imaging tools to locate individual cells on the sampling surface, follow-up measurement via MALDI MS can profile a multitude of single cells in a high-throughput manner. Compared to conventional MS imaging of the entire sample-containing surface, preselection of accurate cell locations for targeted analysis increases selectivity and throughput as well as reduces overall data size. As a result, more informative data sets are produced in less time with fewer resources. Employing this strategy, we present a multistage protocol for optical image-guided, high-throughput single cell analysis by MALDI MS. We outline steps for the isolation and preparation of single cells prior to dispersing on a glass side for optical imaging, automated mapping of imaged cell locations using microMS, preparing the cells for MALDI MS analysis using standards and MALDI matrices, acquisition of mass spectra from mapped individual cells, and data processing and statistical analysis. Images obtained via a range of microscopy modalities can be assessed by the microMS custom image processing software suite to determine location coordinates for cells with defined morphological and / or biochemical characteristics. Depending on the biological model and MS instrumentation used, this strategy can be further enhanced by hyphenation of different imaging modalities, e.g., electron microscopy, for multidimensional sample characterization.

Qualitative and Quantitative Metabolomic Investigation of Single Neurons by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry
P. Nemes, S.S. Rubakhin, J.T. Aerts, J.V. Sweedler, Nat. Protoc. 8, 2013, 783–799.

Abstract: Single-cell mass spectrometry (MS) empowers metabolomic investigations by decreasing analytical dimensions to the size of individual cells and subcellular structures. We describe a protocol for investigating and quantifying metabolites in individual isolated neurons using single-cell capillary electrophoresis (CE) coupled to electrospray ionization (ESI) time-of-flight (TO) MS. The protocol requires ~2 h for sample preparation, neuron isolation and metabolite extraction, and 1 h for metabolic measurement. We used the approach to detect more than 300 distinct compounds in the mass range of typical metabolites in various individual neurons (25–500 mm in diameter) isolated from the sea slug (Aplysia californica) central and rat (Rattus norvegicus) peripheral nervous systems. We found that a subset of identified compounds was sufficient to reveal metabolic differences among freshly isolated neurons of different types and changes in the metabolite profiles of cultured neurons. The protocol can be applied to the characterization of the metabolome in a variety of smaller cells and/or subcellular domains.

A Protease for Middle Down Proteomics
C. Wu, J.C. Tran, L.Zamdborg, K.R. Durbin, M. Li, D.R. Ahlf, B.P. Early, P.M. Thomas, J.V. Sweedler, N.L. Kelleher, Nat. Methods 9, 2012, 822.

Abstract: We developed a method for restricted enzymatic proteolysis using the outer membrane protease T (OmpT) to produce large peptides (>6.3 kDa on average) for mass spectrometry–based proteomics. Using this approach to analyze prefractionated high-mass HeLa proteins, we identified 3,697 unique peptides from 1,038 proteins. We demonstrated the ability of large OmpT peptides to differentiate closely related protein isoforms and to enable the detection of many post-translational modifications.

Neuropeptidomics: Mass Spectrometry-based Qualitative and Quantitative Analysis
P. Yin, X. Hou, E.V. Romanova, J.V. Sweedler, Methods Mol. Biol. 789, 2011, 223–236.

Abstract: Neuropeptidomics refers to a global characterization approach for the investigation of neuropeptides, often under specific physiological conditions. Neuropeptides comprise a complex set of signaling molecules that are involved in regulatory functions and behavioral control in the nervous system. Neuropeptidomics is inherently challenging because neuropeptides are spatially, temporally, and chemically heterogeneous, making them difficult to predict in silico from genomic information. Mature neuropeptides are produced from intricate enzymatic processing of precursor proteins/prohormones via a range of posttranslational modifications, resulting in multiple final peptide products from each prohormone gene. Although there are several methods for targeted peptide studies, mass spectrometry (MS), with its qualitative and quantitative capabilities, is ideally suited to the task. MS provides fast, sensitive, accurate, and high throughput peptidomic analysis of neuropeptides without requiring prior knowledge of the peptide sequences. Aided by liquid chromatography (LC) separations and bioinformatics, MS is quickly becoming a leading technique in neuropeptidomics. This chapter describes several LC-MS analytical methods to identify, characterize, and quantify neuropeptides while emphasizing the sample preparation steps so integral to experimental success.

A Mass Spectrometry Primer for Mass Spectrometry Imaging
S.S. Rubakhin, J.V. Sweedler, Methods Mol. Biol. 656, 2010, 21–49.

Abstract: Mass spectrometry imaging (MSI), a rapidly growing subfield of chemical imaging, employs mass spectrometry (MS) technologies to create single- and multi-dimensional localization maps for a variety of atoms and molecules. Complimentary to other imaging approaches, MSI provides high chemical specificity and broad analyte coverage. This powerful analytical toolset is capable of measuring the distribution of many classes of inorganics, metabolites, proteins, and pharmaceuticals in chemically and structurally complex biological specimens in vivo, in vitro, and in situ. The MSI approaches highlighted in this Methods in Molecular Biology volume provide flexibility of detection, characterization, and identification of multiple known and unknown analytes. The goal of this chapter is to introduce investigators who may be unfamiliar with MS to the basic principles of the mass spectrometric approaches as used in MSI. In addition to guidelines for choosing the most suitable MSI method for specific investigations, cross-references are provided to the chapters in this volume that describe the appropriate experimental protocols. 

Mass Spectrometry Imaging using the Stretched Sample Approach
T.A. Zimmerman, S.S. Rubakhin, J.V. Sweedler, Methods Mol. Biol. 656, 2010, 465–479.

Abstract: Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) can determine tissue localization for a variety of analytes with high sensitivity, chemical specificity, and spatial resolution. MS image quality typically depends on the MALDI matrix application method used, particularly when the matrix solution or powder is applied directly to the tissue surface. Improper matrix application results in spatial redistribution of analytes and reduced MS signal quality. Here we present a stretched sample imaging protocol that removes the dependence of MS image quality on the matrix application processs and improves analyte extraction and sample desalting. First, the tissue sample is placed on a monolayer of solid support beads that are embedded in a hydrophobic membrane. Stretching the membrane fragments the tissue into thousands of nearly single-cell sized islands, with the pieces physically isolated from each other by the membrane. This spatial isolation prevents analyte transfer between beads, allowing for longer exposure of the tissue fragments to the MALDI matrix, thereby improving detectability of small analyte quantities without sacrificing spatial resolution. When using this method to reconstruct chemical images, complications result from non-uniform stretching of the supporting membrane. Addressing this concern, several computational tools enable automated data acquisition at individual bead locations and allow reconstruction of ion images corresponding to the original spatial conformation of the tissue section. Using mouse pituitary, we demonstrate the utility of this stretched imaging technique for characterizing peptide distributions in heterogeneous tissues at nearly single-cell resolution. 

Quantitative Neuroproteomics of the Synapse
D.L. Ramos-Ortolaza, I. Bushlin, N. Abul-Husn, S.P. Annangudi, J.V. Sweedler, L.A. Devi,  Methods Mol. Biol. 615, 2010, 227–246.

Abstract: An emerging way to study neuropsychiatric or neurodegenerative diseases is by performing proteomic analyses of brain tissues. Here, we describe methods used to isolate and identify the proteins associated with a sample of interest, such as the synapse, as well as to compare the levels of proteins in the sample under different conditions. These techniques, involving subcellular fractionation and modern quantitative proteomics using isotopic labels, can be used to understand the organization of neuronal compartments and the regulation of synaptic function under various conditions.

Imaging of Cells and Tissues with Mass Spectrometry: Adding Chemical Information to Imaging
T.A. Zimmerman, E.B. Monroe, K.R. Tucker, S.S. Rubakhin, J.V. Sweedler, Methods Cell Biol. 89, 2008, 361–390.

Abstract: Techniques that map the distribution of compounds in biological tissues can be invaluable in addressing a number of critical questions in biology and medicine. One of the newer methods, mass spectrometric imaging, has enabled investigation of spatial localization for a variety of compounds ranging from atomics to proteins. The ability of mass spectrometry to detect and differentiate a large number of unlabeled compounds makes the approach amenable to the study of complex biological tissues. This chapter focuses on recent advances in the instrumentation and sample preparation protocols that make mass spectrometric imaging of biological samples possible, including strategies for both tissue and single-cell imaging using the following mass spectrometric ionization methods: matrix-assisted laser desorption/ionization, secondary ion, electrospray, and desorption electrospray. 

Characterizing Peptides in Individual Mammalian Cells using Mass Spectrometry
S.S. Rubakhin and J.V. Sweedler, Nat. Protocols 8, 2007, 1987–1997.

Abstract: Cell-to-cell chemical signaling plays multiple roles in coordinating the activity of the functional elements of an organism, with these elements ranging from a three-neuron reflex circuit to the entire animal. In recent years, single-cell mass spectrometry (MS) has enabled the discovery of cell-to-cell signaling molecules from the nervous system of a number of invertebrates. We describe a protocol for analyzing individual cells from rat pituitary using matrix-assisted laser desorption/ionization MS. Each step in the sample preparation process, including cell stabilization, isolation, sample preparation, signal acquisition and data interpretation, is detailed here. Although we employ this method to investigate peptides in individual pituitary cells, it can be adapted to other cell types and even subcellular sections from a range of animals. This protocol allows one to obtain 20–30 individual cell samples and acquire mass spectra from them in a single day.