Enhanced Peptide Detection Toward Single-Neuron Proteomics by Reversed-Phase Fractionation Capillary Electrophoresis Mass Spectrometry
Authors: Peter Nemes
M Chiara Manzini
Pablo Muñoz-LLancao
Camille Lombard-Banek
Sam B Choi
Journal: Journal of the American Society for Mass Spectrometry (2018)
Discipline: CHEMISTRY
Keywords: Bottom-up proteomics; Capillary electrophoresis; Central nervous system; Fractionation; Hippocampus
Affiliations: Department of Chemistry, The George Washington University, Washington, DC, 20052, USA., Institute for Neuroscience, Department of Pharmacology and Physiology, The George Washington University, Washington, DC, 20052, USA., Department of Chemistry, The George Washington University, Washington, DC, 20052, USA. ., Department of Chemistry and Biochemistry, University of Maryland, College Park, MD, 20742, USA. .

Abstract:  The ability to detect peptides and proteins in single cells is vital for understanding cell heterogeneity in the nervous system. Capillary electrophoresis (CE) nanoelectrospray ionization (nanoESI) provides high-resolution mass spectrometry (HRMS) with trace-level sensitivity, but compressed separation during CE challenges protein identification by tandem HRMS with limited MS/MS duty cycle. Here, we supplemented ultrasensitive CE-nanoESI-HRMS with reversed-phase (RP) fractionation to enhance identifications from protein digest amounts that approximate to a few mammalian neurons. An ~1 to 20 μg neuronal protein digest was fractionated on a RP column (ZipTip), and 1 ng to 500 pg of peptides were analyzed by a custom-built CE-HRMS system. Compared with the control (no fractionation), RP fractionation improved CE separation (theoretical plates ~274,000 versus 412,000 maximum, resp.), which enhanced detection sensitivity (2.5-fold higher signal-to-noise ratio), minimized co-isolation spectral interferences during MS/MS, and increased the temporal rate of peptide identification by up to ~57%. From 1 ng of protein digest (&lt;5 neurons), CE with RP fractionation identified 737 protein groups (1,753 peptides), or ~480 protein groups (~1,650 peptides) on average per analysis. The approach was scalable to 500 pg of protein digest (~a single neuron), identifying 225 protein groups (623 peptides) in technical triplicates, or 141 protein groups on average per analysis. Among identified proteins, 101 proteins were products of genes that are known to be transcriptionally active in single neurons during early development of the brain, including those involved in synaptic transmission and plasticity and cytoskeletal organization. Graphical abstract ᅟ. 

Retracted: No

PubPeer comments:
 It has come to my attention that at least one of the main contributors to this paper has been left off. Eric J. Corcoran wrote the first draft of this paper and did a significant part of the experiments. Later, additional experiments were done by Choi, and the text of the manuscript was changed, but Corcoran s name as well as that of another author, Marta Zamarbide, were taken off. There is little left of the original text or figures, but it is sad that the original author was not part of the final submitted version. Could the current authors please comment why they left Corcoran and Zamarbide off? , 
 Some side-by-side comparisons of the published version by Choi et al (on the left) and the draft shared by Corcoran with his PI (on the right). Note that these are mostly not verbatim copied sections, but there are many similarities between the first draft and the final versions. Title pages: ![file] Methods: ![file] ![file] ![file] Figure 2 (published) and Figure 3 (draft) - again, datapoints are not identical, but the original work closely resembles that of the published figure. ![file] , 
 I carefully read the published paper and the draft and I would say these are two different works. Here are the reasons: 1. They use different neuron cell types to perform the experiments; 2. Although they both use two independent methods to purify the digest peptides. The methods they used were different. One of them was UPLC while another was the RP column. They are two different separation methods based on two different mechanisms. 3. I see you highlighted lots of contents are very similar, however, some of them should be similar. For example, people always use DTT to digest the protein disulfide bond and BCA to determine the concentration of proteins. , 
 1. No, cell type was the same. Paper 1 and 2 use enzymatically dissociated hippocampal tissue extracted from embryonic day 16 mouse, which was grown on 24 well plate in medium for 14 days. Resulting in cultured hippocampal neurons, proteins were extracted using the same protocol ending in acetone precipitation of peptides for analysis and storage. Exact same neuron cell type, same breed of animal, same extraction, same purification methods. 2. The methods of separation are the same, with a slight variation explained as follows. The page missing for comparison, is section 3.6 and 3.7 from paper 1, Dr. E. Bik could verify by posting that section. That section denotes the use of CE separation on the fractionated samples from the UPLC. This was methodically produced to compare to extraction done by zip-tip cartridges, also preformed by the graduate student from paper 1. The evidence of zip-tip extraction will be found in the studentu2019s lab notebook and pictures taking during the process. This was obviously a further development on the paper 1 and had not yet been included here but was presented internally to at least eight other individuals. 3. Irrelevant. You made no note on the similarity of the complicated graphs axis of migration time vs. hydrophobicity, and the associated frequency of PSMs from the fractions. Nor did you mention the lack of authorship attributed to E.C. and M.Z. who clearly were involved in the project design, experimentation, and draft of paper 1 and thus of paper 2. 4. You are posting from an anonymous account, so I can not verify if you are an expert, but indeed I AM. , 
 ****Statement from the University of Maryland, College Park and the George Washington University**** In 2019, the University of Maryland, College Park and the George Washington University conducted a joint assessment of an allegation of plagiarism involving the following publication: Choi, S.B., Lombard-Banek, C., Muu00f1oz-LLancao, P., Manzini, M.C., Nemes, P. (2018). Enhanced Peptide Detection Toward Single-Neuron Proteomics by Reversed-Phase Fractionation Capillary Electrophoresis Mass Spectrometry. *Journal of the American Society of Mass Spectrometry*, 29(5), 913-922. doi: 10.1007 s13361-017-1838-1 Specifically, the complaint alleged that the publication contained content from a draft manuscript of another individual who was not given appropriate credit for their contribution in the publication. The joint assessment determined that the studies underlying the publication and the draft manuscript were distinct and different, and did not overlap. The assessment identified critical differences in experimental design, methodology, equipment used to conduct the experiments, underlying data, analysis, and findings. The assessment also found that the other individual was not involved in designing or performing the experiments, collecting or analyzing the data, or drafting or revising the published manuscript. The information presented in response to the allegation was corroborated with supporting evidence. Given this, the initiation of an inquiry under institutional policies was not warranted. Both institutions concurred in this determination, and the individual was notified of the outcome. Due to the restrictions on the information that can be shared regarding such allegations and their review, the institutions will be unable to comment further on this matter. , 
 #3 1. No, cell type was the same. Paper 1 and 2 use enzymatically dissociated hippocampal tissue extracted from embryonic day 16 mouse, which was grown on 24 well plate in medium for 14 days. Resulting in cultured hippocampal neurons, proteins were extracted using the same protocol ending in acetone precipitation of peptides for analysis and storage. Exact same neuron cell type, same breed of animal, same extraction, same purification methods. 2. The methods of separation are the same, with a slight variation explained as follows. The page missing for comparison, is section 3.6 and 3.7 from paper 1, Dr. E. Bik could verify by posting that section. That section denotes the use of CE separation on the fractionated samples from the UPLC. This was methodically produced to compare to extraction done by zip-tip cartridges, also preformed by the graduate student from paper 1. The evidence of zip-tip extraction will be found in the studentu2019s lab notebook and pictures taking during the process. This was obviously a further development on the paper 1 and had not yet been included here but was presented internally to at least eight other individuals. 3. You made no note on the similarity of the complicated graphs axis of migration time vs. hydrophobicity, and the associated frequency of PSMs from the fractions. ,