We developed InSpIon system capable of stable nanoESI spray. Here, K562 cell digests were measured by DDA-MS and DIA-MS with a typical open nanoESI spray system and a spray system inserted spray tip head into an ion transfer tube, and InSpIon system.

We evaluated the impact of surfactants on the recovery of dried peptides. Dried K562 cell digests were dissolved by solutions containing different concentrations of 12 surfactants and measured by DDA-MS and DIA-MS.

This data was utilized to assess the utility of perfusion culture in cultivating spheroids of sarcoma cells. The differences between spheroids cultured with perfusion and without perfusion were analysed through mass spectrometry. The spheroids were fabricated from NCC-UPS4-C1 cell line derived from a patient with undifferentiated pleomorphic sarcoma. We found the different protein expression levels between spheroids cultured with perfusion and without perfusion.

For building a peak assignment algorithm using machine learning, the peptide fragment of extracted proteins from mouse liver was identified. One raw data includes the peptides with heavy- or light-labeled dimethylated lysine resides.

The lauryl maltose neopentyl glycol (LMNG)-assisted sample preparation (LASP) method was adapted for the process of on-beads digestion in co-immunoprecipitation with mass spectrometry (coIP-MS). In addition, to improve peptide recovery, the dried peptides were dissolved in 0.01% decyl maltose neopentyl glycol (DMNG) after desalting at the SDB Stage tip. The adapted LASP method and the lysis of dried peptides with DMNG increased the number of proteins identified in coIP-MS.

Isobaric tags, such as Tandem Mass Tag™ (TMT; Thermo Fisher Scientific), TMTpro™ (Thermo Fisher Scientific), or iTRAQ^® (Sciex), allow reproducible and high-throughput peptide/protein quantification in mass spectrometry-based proteomics. Herein, we introduce a practical protocol for TMT-labeling of peptide samples, with troubleshooting for possible problems and examples of important parameters in measurement by mass spectrometry.

We have developed a lauryl maltose neopentyl glycol (LMNG)-assisted sample preparation (LASP) method for reducing protein and peptide loss. LMNG was removed simply via reversed phase solid-phase extraction. LASP method was applied to the low-input SP3 method and EVOSEP ONE combined with LC-MS/MS. Furthermore, we have established sample preparation method for single cell proteomics based on the LASP method (scpLASP).

For obtaining the structural data of human serum albumin (HSA) in solution using LC-MS analysis, the peptide fragments of HSA were identii ed. The raw data includes the peptides with dimethylated Lysine residues.

We investigated the effect of hypergravity exposure on the proteome profiles of the slow-twitch soleus and fast-twitch extensor digitorum longus muscles of mice. Mice were raised under 3-g (hypergravity) or 1-g (control) conditions, and proteins extracted from the soleus and extensor digitorum longus muscles were subjected to quantitative proteomic analysis using data-dependent acquisition mass spectrometry (DDA-MS). The proteome profiles of the soleus and extensor digitorum longus muscles suggested that biological pathways in different muscle types responded differently to hypergravity exposure.

The effect of microgravity (μ-g) exposure and fructo-oligosaccharide (FOS) ingestion during spaceflight was examined by determining the proteome profiles of the soleus and extensor digitorum longus muscles of mice that were launched to the International Space Station (ISS) and placed in a μ-g or centrifugal artificial 1-g (A1-g) environment for 30 days with or without FOS feeding. Quantitative analysis of proteins extracted from the soleus and extensor digitorum longus muscles suggested that in response to μ-g conditions, the soleus muscle fibers changed from a slow-twitch to a fast-twitch phenotype, and FOS ingestion mildly suppressed metabolic changes and oxidative stress.

We collectively evaluated the results of proteomic analysis of the soleus muscle from three mouse models exposed to different gravity environments (hindlimb unloading, spaceflight, and hypergravity), and identified gravity-responsive muscle proteins.

The microgravity (μ-g) environment during spaceflight can cause severe acute bone loss. To identify gravity-response proteins involved in bone metabolism, we analyzed the proteome of the femur and mandible collected from spaceflight mice raised in a μ-g or artificial 1-gravity (A1-g) environment on the International Space Station in orbit. The bone mass of the femur diaphysis decreased in response to gravity unloading, whereas that of the mandible remained unchanged due to mastication stimuli. Collective evaluation of the dataset of the proteome of the femur and mandible obtained by data-independent acquisition mass spectrometry (DIA-MS) identified the bone proteins that were altered by the μ-g environment and involved in bone loss.

We have developed a novel method for the preparation of proteome samples by direct digestion of methanol-fixed cells with trypsin, named in situ digestion of alcohol-fixed cells for quantitative proteomics (iSDAC). In this method, the cells are fixed in methanol and digested directly with trypsin without solubilizing cells with detergents. The elimination of the detergent removal step allows for easy and rapid sample preparation. This method’s digestion efficiency and reproducibility are comparable to those of commonly used proteome sample preparation methods.

Protein phosphorylation is one of the most major post-translational modifications which is involved in regulation of various cellular functions. The number of phosphorylation sites identified has dramatically increased with the development of phosphoproteome analysis technology by mass spectrometry. The remarkable development of this technology has been supported mainly by the development and improvement of pretreatment methods such as phosphopeptide enrichment, the evolution of mass spectrometers, and the development of data analysis methods and software. In this paper we focus on pretreatment methods and present an example of clinical phosphoproteome analysis with limited sample amounts.

In this study, the differential solubilization method was modified to extract peptides from small amounts of frozen tissue. As a result, we successfully identified a total of 1,535 peptides including 35 bioactive peptides from 135 μg of mouse hypothalamus in three measurements.