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23 Oct 2019

An Accurate Mass Defect-based labeling strategy for Quantitative Proteomics

By nickwiecien

Zhong et al (2019) wrote about their development of an accurate mass-defect based labelling strategy for MS1-centric quantification in a recent Analytical Chemistry paper. 

Specifically, researchers developed 5-plex mass defect N, N-dimethyl leucine (mdDiLeu) tags. These tags have multiple benefits; they can aid in the quantification of biological samples and have increased multiplexing due to the addition of mass difference isotopologues. Additionally, the synthesis of these cost effective tags is straightforward and only requires one reaction step, which can be done in any lab. Also, this mass defect-based labelling strategy is more accurate than isobaric label-based reporter ion quantification, as the latter is impacted by ratio compression.

In this paper, Zhong et al (2019) demonstrate the efficacy of 5-plex mdDiLeu tags for quantitative proteomics by conducting mass spectrometry experiments with these tags on labelled Saccharomyces cerevisiae lysate digest.

Graphical abstract for Zhong et al (2019) depicting the accurate mass-defect labelling strategy used.
16 Oct 2019

Quantification of the Human Pancreatic ECM

By nickwiecien

Ma et al (2019) recently published a paper on the quantification of human pancreatic extracellular matrix proteins in the Journal of Proteome Research.

In this study, researchers characterized the composition of the human pancreatic extracellular matrix (ECM) before and after decellularization. To find the relative quantification of ECM proteins, they used isobaric dimethylated leucine (DiLeu) labeling.

It was important for researchers to look at the ECM of the pancreatic microenvironment as it is essential to pancreatic function– it regulates β cell proliferation, differentiation, and insulin secretion.

As a result of decellularization, and through quantitative proteomic analysis, most cellular proteins were removed while matrisome proteins remained. This process generated a large data set of matrisome proteins from a single tissue type. 

Researchers then quantified the distinct expression of ECM proteins, comparing adult and fetal pancreas ECM. This revealed a correlation between matrix composition and postnatal β cell maturation.

Overall, the results of this study sheds light on the prospect of bioengineering a pancreas. Additionally, the study demonstrates the roles that matrisome proteins have in postnatal β cell maturation.

Graphical abstract for Ma et al (2019), depicting native and decelled pancreatic extracellular matrix proteins, sample preparation technique, and LFQ
09 Oct 2019

Fixed mass-to-charge ratio scan ranges generates more MS/MS scans than standard approaches

By nickwiecien

Trujillo et al (2019) published an article on maximizing tandem mass spectrometry acquisition rates for shotgun proteomics in a recent issue of Analytical Chemistry.

While advances in mass spectrometry (MS/MS) have lead to increased performance in shotgun proteomics experiments, ion trap scan duration is highly variable and often depends on the mass of the precursor.

Looking into this variability, the authors compared the performance of various static mass-to-charge ratio scan ranges for ion trap MS/MS acquisition to conventional dynamic mass-to-charge ratio scan ranges. Compared to the standard dynamic approach, the fixed mass-to-charge ratio scan range generated 12% more MS/MS scans and identified more unique peptides.

Graphical abstract for Trujillo et al (2019) depicting a graph titled "Increase # MS/MS collected." We see that as the maximum ion injection time decreases, the number of MS/MS collected increases. Additionally, as the m/z scan range increases, the # of MS/MS increases as well.
02 Oct 2019

Acute-Phase proteins differ in mice with and without prostate inflammation

By nickwiecien

L. Hao et al conducted a quantitative proteomic analysis on induced prostate inflammation in mice and published a paper in the American Journal of Physiology-Renal Physiology.

Researchers compared the quantitative proteomic analysis of urine from mice with and without prostate-specific inflammation. Prostate inflammation as it is a key symptom of many different prostate conditions, such as infection and cancer, and therefore by doing so one gains a better understanding of disease mechanisms.

Researchers induced prostate-specific inflammation by conditional prostate epithelial IL-1β expression. Next, they ran urine sample tests and quantified urinary proteins. L. Hao et al found that different levels of acute-phase response proteins (proteins which have plasma concentrations that increase or decrease in response to inflammation) were represented between mice with and without prostate inflammation; these were haptoglobin, inter-α-trypsin inhibitor, and α1-antitrypsin 1-1.

Mass-spectrometry-based quantitative urinary proteomics is an important and powerful method for discovering biomarkers and uncovering molecular urological mechanisms.

Graphical abstract for Hao et al, depicting the quantitative proteomic analysis of mice urine.
25 Sep 2019

Polo-like kinase 4 maintains integrity of centriolar satellites

By nickwiecien

RA Denu et al recently published a paper on the importance of polo-like kinase 4 for centriolar satellite integrity in the Journal of Biological Chemistry.

Polo-like kinase 4 (PLK4), a Ser/Hr protein kinase, is the “master regulator” of centriole duplication and can also play a role in centrosome function. Centrioles are cell organelles which are responsible for cell division. In addition, centrioles are housed in other organelles, called centrosomes. 
This study was an attempt to identify additional proteins regulated by PLK4. To do this, scientists generated an RPE-1 human cell line and genetically engineered analog sensitive PLK4As.

Scientists found that Ser-78 is important for maintaining the integrity of centriolar satellites, as this is where PLK4 phosphorylates CEP131. Ser-78 in centrosomal protein 131 is a direct substrate of PLK4.

Another finding is that inhibiting PLK4 or using a nonphosphorylatable CEP131 tended to result in “dispersed” centriolar satellites.

18 Sep 2019

CZE and Mass Spectrometry Leads to Considerable Phosphopeptide Identification

By nickwiecien

Zhang et al demonstrated the importance and utility of Single-Shot Capillary Zone Electrophoresis in the Journal of Proteome Research.

Capillary zone electrophoresis (CZE) is a practical tool in exploring and interpreting post-translational modifications in proteins. To examine the usefulness of single-shot CZE with mass spectrometry through the analysis of phosphoproteomics, researchers used CZE separations with the Orbitrap Fusion Lumos Tribrid platform, and used a linear-polyacrylamide-coated capillary with low electroosmotic flow for separation.

Researchers found that larger injection volumes led to broader peaks and less phosphopeptide identifications. Additionally, in this single-shot phosphoproteome analysis, researchers found 4405 phosphopeptides out of an original 220 ng enriched phosphopeptides from a mouse brain.

Data for this study is available in the ProteomeXchange with identified PXD012888.

Graphical abstract for Zhang et al. This graph shows how Base Peak Intensity compares to Migration Time (in minutes). It appears that base peak intensity is higher during the 38 minute mark, the 63 minute mark, and the 65 minute mark.
11 Sep 2019

NCQBCS Scientists Publish on Essential Phosphatase Pptc7

By nickwiecien

Niemi et al analyzed a Pptc7 matrix phosphatase in mice in a recent issue of Nature Communications.

This paper addressed the functionality of phosphorylation in mitochondrial proteins. While mitochondrial proteins tend to have a lot of phosphorylation, it is also possible that protein dephosphorylation (the opposite process) may be significant in controlling various mitochondrial processes.

To test this, researchers deleted the matrix phosphatase Pptc7 from mice using the CRISPR-Cas9. As a result, mice were born with normal transcript levels but less mitochondria and protein in their tissues. They also had more phosphorylation in certain mitochondrial proteins. These mice developed hypoketonic hypoglycemia, had higher levels of acylcarnitines and serum lactate, and died shortly after being born. 

Analyzing this data, researchers pinpointed that the protein translocase complex subunit Timm50 is probably a Pptc7 substrate whose phosphorylation lowers import activity.  This data also demonstrates that Pptc7 is necessary for healthy mammalian mitochondrial processes, such as metabolism, and biogenesis after birth. 

04 Sep 2019

Brademan Paper on the Interactive Peptide Spectral Annotator

By nickwiecien

Brademan et al unveiled the Interactive Peptide Spectral Annotator (IPSA), in a recent issue of Molecular and Cellular Proteomics.

The IPSA is an interactive and easily-accessible web-based annotator that can be used to conceptualize and characterize peptides with mass spectra. This tool, which can visualize peptides collected from different experimental and instrumental sources, has a variety of purposes including creating figures for publication, annotating spectra for negative-mode ionization and the like.

The IPSA can be accessed through this link:
http://www.interactivepeptidespectralannotator.com

21 May 2019

McKetney Publishes “Proteomic Atlas of the Human Brain in Alzheimer’s Disease”

By nickwiecien

Justin McKetney et. al. recently published a paper on neurodegenerative diseases of the brain, such as Alzheimer’s disease (AD). Using state-of-the-art mass spectrometry, the group identified a core brain proteome where substantial differences were identified between previous proteomic studies of mature adult brains and their aged cohort. These findings suggest considerable value in examining specifically the brain proteome of aged human populations and can serve as a guide for how specific regions of the brain are affected by aging and neurodegeneration.

Graphical abstract for McKetney et al depicting areas of the brain studies, and methods used for creating a proteomic atlas: protein extraction, fractionation, chromatography, tandem MS, and Database search.
28 Jan 2019

Article selected for Journal of Biological Chemistry 2018 Collection

By nickwiecien

The Mitok/Coon/Attie article “Islet proteomics reveals genetic variation in dopamine production resulting in altered insulin secretion” was selected as the representative ‘Genomics and proteomics’ article for the Journal of Biological Chemistry 2018 Retrospective Collection called “The year in JBC: 2018.” When choosing the representative articles, the journal editors considered hundreds of papers to come up with what they felt best represented the exciting advances reported in JBC last year. This special issue can be found at http://www.jbc.org/site/vi/.

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