December 13, 2011
Dear Editor,
Re: JCA-11-1997 “Total glycosaminoglycan disaccharide analysis by ultra-performance liquid
chromatography-mass spectrometry”
We are pleased that the expert reviewers think our work is of potential interest to the readership of
Journal of Chromatography A and you would consider a revised manuscript for publication. We are
grateful to the reviewers for their evaluation and the critical comments. On the attached pages we
respond to these comments and revise our manuscript wherever possible to address their concerns.
I trust that our responses and the revisions will make the manuscript acceptable for publication in
Journal of Chromatography A.
Yours sincerely
Robert J. Linhardt Ph.D.
Professor of Chemistry and Chemical Biology,
Biology, and Chemical and Biological Engineering
Rensselaer Polytechnic Institute, Biotech Center 4005,
110 8th Street,
Troy, NY 12180-3590, USA
Reviewer #1:
This reviewer thinks that our “manuscript illustrates a UPLC-MS separation and identification of total
GAGs disaccharides produced from heparin/heparan sulfate, chondroitin sulfate/dermatan sulfate and
hyaluronan.” And this reviewer also appreciates that “the quality of this study is good”. But the reviewer
suggested that “the novelty of this research and its utility needs to be better clarified for the readers of J.
Chrom. A”.
As the reviewer mentioned, several paper have reported that the labeling GAG-derived disaccharides
with AMAC fluorophore were separated and analyzed by reverse-phase (RP)-HPLC. We have reviewed
and cited these papers in the introduction section of our manuscript. These studies only achieved
separations for mixtures of 8 or 12 HS/HP disaccharides (J.A. Deakin, Glycobiology 2008; M.
Ambrosius, J. Chromatogr. A 2008; F. Galeotti, Anal. Chem. 2011), or mixtures of 9 CS/DS/HA
disaccharides (J.A. Deakin, Glycobiology 2008; M. Ambrosius, J. Chromatogr. A 2008, Volpi, Anal.
Biochem. 2010). This is in contrast to the separation of a mixture of 17 HP/HS/CS/DS/HA disaccharides
described in our study. We also point out in our manuscript that “all the current methods still require the
separate recovery of HS/HP disaccharides and CS/DS disaccharides from multiple digestion steps,
followed by separate and distinctly different analyses affording a very time-consuming and complicated
analytical process. Moreover, the resulting data from multiple analyses are often difficult to compare
and can provide inaccurate calculations of the ratio of HS/HP and CS/DS present in a sample”.
Nevertheless, the analysis of multiple classes of GAG disaccharides still has not previously been
reported. Moreover, we describe a simplified experimental method and shortened analysis times.
UPLC takes advantage of technological strides made in resolution, peak capacity, sensitivity, efficiency
and speed of analysis. It has been widely used in many analytical fields as advanced technology. Many
publications have reported the advantages and applications of UPLC. In our previous study, which
compares UPLC to HPLC (Yang et al., Anal. Biochem. 2011), we found that both UPLC and traditional
HPLC column show good separation for analysis of eight heparin/HS disaccharides standards and
pharmaceutical heparin samples. But in the analysis of “real” biological samples from cells and tissues,
UPLC significantly improves separation, sensitivity, and analysis speed without being subject to
interference from proteins, peptides and other sample impurities were obtained by using UPLC column
than traditional HPLC column. Therefore, we used UPLC in this work and separated the 17 different
GAG-derived disaccharides with stable retention times, high sensitivity and excellent calibration curves.
We have added text to clarify the novelty and utility of our research.
This reviewer has some specific comments which we address:
1.
Introduction. The sentence "It is biosynthesized .. 2-O-sulfo groups" should be shortened.
This sentence has been shortened.
2.
Introduction. "...afford eight HP/HS-derived disaccharides." Indeed heparin lyase treatment
affords 12 unsaturated disaccharides.
This statement has been revised.
3.
Introduction. The sentence "Liquid chromatography... For disaccharide analysis" should be
shortened.
This sentence has been shortened.
4.
Experiment. 5 microg of samples has been treated with lyases. How this amount was determined?
The amount was determined using micro carbazole assay. We digested about 5 μg of GAG samples, but
only injected 0.5 ng-100 ng to do LC/MS analysis in order to the amount of each disaccharide within the
calibration curve. We are sorry to have confused the readers. We have added this information in the
experimental section.
5. Experiment. Due to the use of various lyases, the effective capacity to degrade heparin/HS,
CS/CS/HA and the recovery of disaccharides of different GAGs should be performed by recovery studies.
The recovery of GAGs from tissues has been previously well described (Turnbull et al 2009). We are
currently working on improved methods of the rapid micro recovery of GAGs from different cell and
tissue samples and assessing recovery efficiency but this is well beyond the scope of the current study.
6. Experiment. Heparin/HS are composed of twelve unsaturated disaccharides, four of these having
free amino group not considered in this study. This should be performed or discussed inside the text.
This is now described in our revised manuscript.
7.
Results and Discussion. The first sentence is a repetition.
This sentence has been removed.
8.
Results and Discussion. Figure 1 is not useful as it is well known.
We have modified/condensed but have retained Figure 1 as we believe that it is helpful for the nonexpert reader to understand the structure of the molecules being separated.
9.
"Optimization of mobile phase" section should be strongly reduced. Tables S1 and S2 are not useful.
The ammonium acetate concentration is an important factor required for the separation of the 17
disaccharides. It also provides important information on the relationship between ammonium acetate
concentration and each disaccharides retention time shift. All optimization of this separation depends on
these information. Tables S1 and S2 showed all linear equations, their sensitivity and detection range.
All quantification results are based on these data. We put these figures in the supplementary materials
section as we believe that these figures, while important are not critical to this manuscript. We believe
that these supporting information will be useful to scientists trying to further improve discaccharide
separations or using supports from different manufacturers.
10.
Results and Discussion. Sections 3.3 and 3.4 are too long with no useful informations.
These sections have been shortened to include only the most useful and important information needed
for quantitative disaccharide analysis. Additional information has also been added in response to the
reviewer’s comment #11.
11. Results and Discussion. All quantitative data about natural extracts should be compared with
those present in previously published papers or with those obtained by conventional published methods.
There are different disaccharide samples isolated from the GAGs of different species and tissues and/or
the same species of different ages. The GAG composition of camel (1 and 2 yr) livers and urine come
from different age animals than previously examined and thus the composition cannot be directly
compared. Our previous publication on camel liver only studies the HS/HP disaccharides and do not
examine the CS/DS disaccharides. In the current study, we give the total GAG composition. If we only
compare HS/HP disaccharides portion of our analysis, the current results using RP-UPLC are consistent
to our previous results, which relied on RPIP-HPLC. CHO cell GAGs analyzed by RP-UPLC are
consistent to our previous results, which relied on RPIP-HPLC. We had added several sentences to
clarify this point.
Reviewer #2:
We thank the reviewer for indicating that our manuscript is “a well designed study that addresses a
significant problem, the total analysis of gags in biologic samples by rp-hplc-ms methodology”. This
reviewer also states that our “results are well validated and this reviewer suggest publication of this
study.”
Minor issues raised by Reviewer 2 are:
1) Authors should take care of several points in the text where for example it is given that gags are
located at the cell surface (see abstract) without to note that they present at both extracellular and
cellular (surface and intracellular) levels.
We have corrected this statement in our revised manuscript.
2) It should be given in several places that certain procedures are for CS/DS and other for HP/HS and
avoiding to refer total gags, it confuses the non-expert on the subject. Therefore, it is suggested that
author have a final reading and replace the text in several places before publication.
We have now carefully clarified these sections in our revised manuscript.