| Title |
Minimizing Carry-Over in an Online Pepsin Digestion System used for the H/D Exchange Mass Spectrometric Analysis of an IgG1 Monoclonal Antibody
|
|---|---|
| Published in |
Journal of the American Society for Mass Spectrometry, September 2012
|
| DOI | 10.1007/s13361-012-0485-9 |
| Pubmed ID | |
| Authors |
Ranajoy Majumdar, Prakash Manikwar, John M. Hickey, Jayant Arora, C. Russell Middaugh, David B. Volkin, David D. Weis |
| Abstract |
Chromatographic carry-over can severely distort measurements of amide H/D exchange in proteins analyzed by LC/MS. In this work, we explored the origin of carry-over in the online digestion of an IgG1 monoclonal antibody using an immobilized pepsin column under quenched H/D exchange conditions (pH 2.5, 0 °C). From a consensus list of 169 different peptides consistently detected during digestion of this large, ~150 kDa protein, approximately 30% of the peptic peptides exhibited carry-over. The majority of carry-over originates from the online digestion. Carry-over can be substantially decreased by washing the online digestion flow-path and pepsin column with two wash cocktails: [acetonitrile (5%)/isopropanol (5%)/acetic acid (20%) in water] and [2 M guanidine hydrochloride in 100 mM phosphate buffer pH 2.5]. Extended use of this two-step washing procedure does not adversely affect the specificity or activity of the immobilized pepsin column. The results suggest that although the mechanism of carry-over appears to be chemical in nature, and not hydrodynamic, carry-over cannot be attributed to a single factor such as mass, abundance, pI, or hydrophobicity of the peptides. |
X Demographics
The data shown below were collected from the profiles of 2 X users who shared this research output. Click here to find out more about how the information was compiled.
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| United States | 1 | 50% |
| Unknown | 1 | 50% |
Demographic breakdown
| Type | Count | As % |
|---|---|---|
| Scientists | 2 | 100% |
Mendeley readers
The data shown below were compiled from readership statistics for 69 Mendeley readers of this research output. Click here to see the associated Mendeley record.
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Denmark | 1 | 1% |
| Unknown | 68 | 99% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Student > Ph. D. Student | 24 | 35% |
| Researcher | 11 | 16% |
| Student > Master | 8 | 12% |
| Student > Doctoral Student | 4 | 6% |
| Other | 4 | 6% |
| Other | 6 | 9% |
| Unknown | 12 | 17% |
| Readers by discipline | Count | As % |
|---|---|---|
| Chemistry | 17 | 25% |
| Biochemistry, Genetics and Molecular Biology | 13 | 19% |
| Agricultural and Biological Sciences | 9 | 13% |
| Medicine and Dentistry | 5 | 7% |
| Pharmacology, Toxicology and Pharmaceutical Science | 4 | 6% |
| Other | 3 | 4% |
| Unknown | 18 | 26% |
Attention Score in Context
This research output has an Altmetric Attention Score of 2. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 01 March 2013.
All research outputs
#15,740,207
of 25,374,647 outputs
Outputs from Journal of the American Society for Mass Spectrometry
#2,364
of 3,834 outputs
Outputs of similar age
#113,607
of 188,919 outputs
Outputs of similar age from Journal of the American Society for Mass Spectrometry
#14
of 50 outputs
Altmetric has tracked 25,374,647 research outputs across all sources so far. This one is in the 37th percentile – i.e., 37% of other outputs scored the same or lower than it.
So far Altmetric has tracked 3,834 research outputs from this source. They receive a mean Attention Score of 3.8. This one is in the 36th percentile – i.e., 36% of its peers scored the same or lower than it.
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