Publications by authors named "Joseph A Loo"

300 Publications

ClipsMS: An Algorithm for Analyzing Internal Fragments Resulting from Top-Down Mass Spectrometry.

J Proteome Res 2021 Apr 2;20(4):1928-1935. Epub 2021 Mar 2.

Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, California 90095, United States.

Top-down mass spectrometry (TD-MS) of peptides and proteins results in product ions that can be correlated to polypeptide sequence. Fragments can either be terminal fragments, which contain either the N- or the C-terminus, or internal fragments that contain neither termini. Normally, only terminal fragments are assigned due to the computational difficulties of assigning internal fragments. Here we describe ClipsMS, an algorithm that can assign both terminal and internal fragments generated by top-down MS fragmentation. Further, ClipsMS can be used to locate various modifications on the protein sequence. Using ClipsMS to assign TD-MS generated product ions, we demonstrate that for apo-myoglobin, the inclusion of internal fragments increases the sequence coverage up to 78%. Interestingly, many internal fragments cover complementary regions to the terminal fragments that enhance the information that is extracted from a single top-down mass spectrum. Analysis of oxidized apo-myoglobin using terminal and internal fragment matching by ClipsMS confirmed the locations of oxidation sites on the two methionine residues. Internal fragments can be beneficial for top-down protein fragmentation analysis, and ClipsMS can be a valuable tool for assigning both terminal and internal fragments present in a top-down mass spectrum. Data are available via the MassIVE community resource with the identifiers MSV000086788 and MSV000086789.
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http://dx.doi.org/10.1021/acs.jproteome.0c00952DOI Listing
April 2021

Farewell and Hello: Associate Editor Changes.

Authors:
Joseph A Loo

J Am Soc Mass Spectrom 2021 Mar 12;32(3):615. Epub 2021 Feb 12.

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http://dx.doi.org/10.1021/jasms.1c00052DOI Listing
March 2021

In Pursuit of Happiness.

Authors:
Joseph A Loo

J Am Soc Mass Spectrom 2021 Jan;32(1):1-2

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http://dx.doi.org/10.1021/jasms.0c00411DOI Listing
January 2021

Unequivocal Identification of Aspartic Acid and Aspartic Acid by MALDI-TOF/TOF: From Peptide Standards to a Therapeutic Antibody.

J Am Soc Mass Spectrom 2021 Jan 3. Epub 2021 Jan 3.

Amgen Research, Discovery Attribute Sciences, Amgen, Inc., Thousand Oaks, California 91320, United States.

Aspartic acid (Asp) to aspartic acid (Asp) isomerization in therapeutic monoclonal antibodies (mAbs) and other biotherapeutics is a critical quality attribute (CQA) that requires careful control and monitoring during the drug discovery and production processes. The unwanted formation of Asp within biotherapeutics and resultant structural changes in the peptide backbone may negatively impact the efficacy, potency, and safety of the molecule or become immunogenic, especially if the isomerization occurs within the mAb complementarity determining region (CDR). Herein we describe a MALDI-TOF/TOF mass spectrometry method that affords unequivocal identification of the presence and the exact position of the Asp residue(s) in peptide standards ranging in size from a tripeptide to a docosapeptide (22 residues). In general, the peptide bond immediately N-terminal to the Asp residue is more susceptible to MALDI-TOF/TOF fragmentation than its unmodified counterpart. In some of the peptides evaluated in this study, fragmentation of the peptide bond C-terminal to the Asp residue (the aspartate effect) is also enhanced when compared to the control. Relative quantification by MALDI-TOF/TOF of this chemical modification is dependent upon a successful reversed-phase HPLC (rpHPLC) separation of the control and modified peptides. This method has also been validated on a therapeutic mAb that contains a well-documented Asp residue in the heavy chain CDR3 after forced degradation. Moreover, we also demonstrate that higher energy C-trap dissociation of only the singly charged species, and not the multiply charged form, of the Asp containing peptide, separated by rpHPLC, results in LC-MS/MS fragmentation that is highly consistent to that of MALDI-TOF/TOF.
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http://dx.doi.org/10.1021/jasms.0c00370DOI Listing
January 2021

I Am Woman, Hear Me Roar (in Science).

Authors:
Joseph A Loo

J Am Soc Mass Spectrom 2020 Nov;31(11):2236

Department of Chemistry & Biochemistry and Department of Biological Chemistry, David Geffen School of Medicine at the University of California, Los Angeles.

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http://dx.doi.org/10.1021/jasms.0c00383DOI Listing
November 2020

Leveraging Immonium Ions for Targeting Acyl-Lysine Modifications in Proteomic Datasets.

Proteomics 2021 Feb 25;21(3-4):e2000111. Epub 2020 Sep 25.

Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, 90095, USA.

Acyl modifications vary greatly in terms of elemental composition and site of protein modification. Developing methods to identify acyl modifications more confidently can help to assess the scope of these modifications in large proteomic datasets. The utility of acyl-lysine immonium ions is analyzed for identifying the modifications in proteomic datasets. It is demonstrated that the cyclized immonium ion is a strong indicator of acyl-lysine presence when its rank or relative abundance compared to other ions within a spectrum is considered. Utilizing a stepped collision energy method in a shotgun experiment highlights the immonium ion. By implementing an analysis that accounted for features within each MS spectrum, the method clearly identifies peptides with short chain acyl-lysine modifications from complex lysates. Immonium ions can also be used to validate novel acyl modifications; in this study, the first examples of 3-hydroxylpimelyl-lysine modifications are reported and they are validated using immonium ions. Overall these results solidify the use of the immonium ion as a marker for acyl-lysine modifications in complex proteomic datasets.
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http://dx.doi.org/10.1002/pmic.202000111DOI Listing
February 2021

To Improve Is To Change···Again.

Authors:
Joseph A Loo

J Am Soc Mass Spectrom 2020 Jan;31(1)

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http://dx.doi.org/10.1021/jasms.9b00126DOI Listing
January 2020

For the Love of Analytical Instruments.

Authors:
Joseph A Loo

J Am Soc Mass Spectrom 2020 Sep;31(9):1773-1774

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http://dx.doi.org/10.1021/jasms.0c00304DOI Listing
September 2020

Interlaboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry.

J Am Soc Mass Spectrom 2020 Sep 19;31(9):1783-1802. Epub 2020 Aug 19.

Pacific Northwest National Laboratory, Richland, Washington 99354, United States.

The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product's primary structure accurately and reproducibly. The major objective of the present study is to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up (BU) approaches with regard to confirming protein integrity, sequencing variable domains, avoiding artifacts, and revealing modifications and their locations. We also aim to gather information on the common TD/MD MS methods and practices in the field. A panel of three mAbs was selected and centrally provided to 20 laboratories worldwide for the analysis: Sigma mAb standard (SiLuLite), NIST mAb standard, and the therapeutic mAb Herceptin (trastuzumab). Various MS instrument platforms and ion dissociation techniques were employed. The present study confirms that TD/MD MS tools are available in laboratories worldwide and provide complementary information to the BU approach that can be crucial for comprehensive mAb characterization. The current limitations, as well as possible solutions to overcome them, are also outlined. A primary limitation revealed by the results of the present study is that the expert knowledge in both experiment and data analysis is indispensable to practice TD/MD MS.
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http://dx.doi.org/10.1021/jasms.0c00036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7539639PMC
September 2020

Internal Fragments Generated by Electron Ionization Dissociation Enhance Protein Top-Down Mass Spectrometry.

J Am Soc Mass Spectrom 2020 Sep 17;31(9):1896-1902. Epub 2020 Aug 17.

Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California 90095, United States.

Top-down proteomics by mass spectrometry (MS) involves the mass measurement of an intact protein followed by subsequent activation of the protein to generate product ions. Electron-based fragmentation methods like electron capture dissociation and electron transfer dissociation are widely used for these types of analyses. Recently, electron ionization dissociation (EID), which utilizes higher energy electrons (>20 eV) has been suggested to be more efficient for top-down protein fragmentation compared to other electron-based dissociation methods. Here, we demonstrate that the use of EID enhances protein fragmentation and subsequent detection of protein fragments. Protein product ions can form by either single cleavage events, resulting in terminal fragments containing the C-terminus or N-terminus of the protein, or by multiple cleavage events to give rise to internal fragments that include neither the C-terminus nor the N-terminus of the protein. Conventionally, internal fragments have been disregarded, as reliable assignments of these fragments were limited. Here, we demonstrate that internal fragments generated by EID can account for ∼20-40% of the mass spectral signals detected by top-down EID-MS experiments. By including internal fragments, the extent of the protein sequence that can be explained from a single tandem mass spectrum increases from ∼50 to ∼99% for 29 kDa carbonic anhydrase II and 8.6 kDa ubiquitin. When searching for internal fragments during data analysis, previously unassigned peaks can be readily and accurately assigned to confirm a given protein sequence and to enhance the utility of top-down protein sequencing experiments.
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http://dx.doi.org/10.1021/jasms.0c00160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7485267PMC
September 2020

Science Moves Forward When We Work Together.

Authors:
Joseph A Loo

J Am Soc Mass Spectrom 2020 Jul 23;31(7):1324-1325. Epub 2020 Jun 23.

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http://dx.doi.org/10.1021/jasms.0c00218DOI Listing
July 2020