Urea spontaneously dissociates to form cyanate and ammonia in aqueous solutions [ 14 ]. Based on this knowledge, strategies have been employed to protect peptides from urea carbamylation by either reducing the generation of cyanates or removing active cyanates from solution.
For example, most proteomic procedures involving the use of urea solution suggest that it should be freshly prepared and further deionized prior to use to reduce cyanate from the beginning of the sample preparation procedure. Some proteomic protocols indicate that either the urea needs to be removed from the sample before digestion, or the sample should be maintained at low temperature to reduce the decomposition rate of urea [ 14 ; 15 ].
Acidifying the sample is another method to drive the equilibrium to favor urea over the formation of isocyanic acid [ 14 ; 16 ]. These strategies, however, require a long handling time and may not be compatible with many enzymatic digestion procedures.
Additionally, removing urea before protein digestion may result in the precipitation of many readily denatured proteins. Numerous amino-containing reagents, such as methylamine, ethanolamine, ethylenediamine, Tris-HCl and 1,2-ethylene diamine, have been suggested for use as cyanate scavengers for protein digestion in urea solution [ 9 ; 17 ].
These reagents work by competing with peptides for cyanates, thereby minimizing protein or peptide carbamylation.
More importantly, NH 4 HCO 3 solution is a commonly used buffer in many enzymatic digestion protocols and therefore is very applicable for proteomic research. Standard peptides angiotensin and neurotensin, standard protein bovine fetuin, human sera frozen liquid , tris 2-Carboxyethyl phosphine TCEP , iodoacetamide, ammonium bicarbonate, ammonium acetate, triethylammonium bicarbonate buffers and Tris-HCl buffer pH 7. Louis, Missouri,.
Sequencing-grade modified trypsin was purchased from Promega Corp. Madison, WI. C18 zip-tips were purchased from Millipore Bedford, MA. Samples were eluted from the C18 tips with 1. The samples were diluted 5-fold with the respective buffers as mentioned above final concentration of urea was 1.
Two microliters of human serum Sigma was diluted fold with 8M urea in one of the following buffers: PB, pH8; 0. The samples were desalted by employing 1cc C18 columns and dried in a SpeedVac.
The dried peptides were resuspended in 0. Spectra were acquired in the reflector mode. The relative carbamylation ratio using the peak area of the carbamylated peptide divided by the peak area of non-carbamylated peptides was used for quantification of the carbamylated peptides.
The spray voltage was set at 2. Charge state screening was enabled to reject unassigned and singly charged ions. A dynamic exclusion time of 35 sec was used to discriminate against previously selected ions. The database search parameters were set as follows: two missed protease cleavage sites were allowed for trypsin digests with 10 ppm precursor mass tolerance and 0.
However, because carbamylation only occurs sporadically at the arginine side chain [ 15 ], the potential carbamylation site on this peptide is its N-terminal residue. Neurotensin contains a pyroglutamic acid residue at its N-terminus; hence, it does not have an active amino group at its N-terminus that can be carbamylated.
Although it contains one lysine residue and two arginine residues as potential carbamylation sites, carbamylation would mainly occur at the lysine residue. Both peptides were incubated in 1. Standard peptides used for peptide carbamylation assay and the peptide masses of their unmodified and carbamylated form. To determine the extent of carbamylation of the N-terminal amino group and lysine amino group in urea with different buffers, we calculated the relative carbamylation ratio using the peak area of the carbamylated peptide divided by the peak area of non-carbamylated peptides.
The relative ratio of carbamylation does not reflect the percent of carbamylation of a specific peptide since the ionization efficiencies of the carbamylated and non-carbamylated forms of the same peptide are different.
However, the relative carbamylation ratio provides a convenient means of measuring the extent of carbamylation of the same peptide in different conditions. The relative carbamylation ratios of both peptides in different buffers are shown in Figure 1. For angiotensin, which contains an amino group at its N-terminus, the relative ratio of carbamylation is 0.
For neurotensin, which does not contain an amino group at its N-terminus, the carbamylation mainly occurs at the lysine residues with relative carbamylation ratios of 0.
The relative carbamylation ratio is reduced to 0. The NH 4 HCO 3 buffer supplied the highest protection to peptides against carbamylation among all the buffers that we tested. Inhibition of peptide carbamylation by different buffers. A Carbamylation of angiotensin peptide at its N-terminus in different buffers with urea.
B Carbamylation of neurotensin at the side chain of Lys in different buffers with urea. C Relative carbamylation of angiotensin and neurotensin in different buffers with urea. The results of relative carbamylation ratio are shown in Figure 1D. The general tendency observed for both peptides was that the relative ratio of carbamylation decreased with increased NH 4 HCO 3 concentrations. The relative peptide carbamylation ratio was only 0. The effect of temperature and urea concentration on carbamylation inhibition by NH 4 HCO 3 solution was also investigated.
However, the relative carbamylation ratio of angiotensin and neurotensin reached The urea concentration was another very important factor that affected the carbamylation level of peptide. The data showed a general trend that the carbamylation of peptides would continue increasing with increased urea concentrations, even in the presence of 0.
Considering all the factors listed above, it was concluded that the carbamylation at both the N-termini and lysine side chains of peptides could be avoided in 1M NH 4 HCO 3 buffer with 1. To investigate the effect of different buffers on protein digestion, a standard protein bovine fetuin was selected for trypsin digestion and carbamylation measurement.
The undetected peptides could contain glycosylation 2 peptides or other unknown modifications 3 peptides. To further assess the protective effect of NH 4 HCO 3 buffer on peptide carbamylation during protein digestion, six tryptic peptides from fetuin were selected for further analysis to determine the relative carbamylation ratio in different buffers based on N-terminal carbamylation, lysine carbamylation of peptides after tryptic digestion, and lysine carbamylation at the protein level causing a missed cleavage of tryptic peptides at the carbamylated lysine Table 2.
Two of the tryptic peptides contain internal N-terminal amino groups and arginine residues at their C-termini peptide 1 and 2 in Table 2 , another two contain internal N-terminal amino groups and lysine residues at their C-termini peptide 3 and 4 in Table 2 , while the remaining two contain carbamylated lysine residues at the missed cleaved lysine residues, internal N-terminal amino groups, and arginine residues at the C-termini of the tryptic peptides peptide 5 and 6 in Table 2.
The relative carbamylation ratios at the N-terminal of peptide 1 and 2 were 0. However, the total carbamylation ratios of the double carbamylation tryptic peptides containing both lysine residues at their C-terminus and internal N-terminal amino groups peptide 3 and 4 were 0. These data indicated that the carbamylation at both N-terminal and lysine residues of peptides could be efficiently inhibited in 1M NH 4 HCO 3 buffer. The two selected peptides with internal missed cleaved lysines were not detected by MALDI-TOF-MS, which suggested that the carbamylation that occurs on the protein level during the denaturation, reduction and alkylation processes was negligible, and the trypsin digestion was equally complete in all four buffers.
The results showed that NH 4 HCO 3 buffer provided the best protection against carbamylation on both protein and peptide levels, and 1M NH 4 HCO 3 almost completely inhibited protein and peptide carbamylation.
Relative carbamylation by protein digestion in different buffers with urea. Bovine fetuin was denatured, reduced, alkylated, and digested in different buffers 0. Six bovine fetuin tryptic peptides that were selected for peptide carbamylation analysis. As described above and shown in Figure 2 , the 1M NH 4 HCO 3 buffer provides the best protection against protein and peptide carbamylation during protease digestion.
When 1M NH 4 HCO 3 buffer with urea was used to denature, reduce, alkylate and digest serum proteins, followed by mass spectrometric analysis of tryptic peptides, the database search results showed that carbamylation was almost completely avoided only one peptide was identified in its carbamylated form.
In contrast, when serum proteins were digested in other buffers, 6. The results from the proteomic analysis of tryptic peptides by tandem mass spectrometry further verified that the majority of the carbamylation occurred at the N-terminal amino groups of peptides Fig.
The 1M NH 4 HCO 3 buffer also did not show any adverse effects on trypsin digestion based on the number of identified peptides and missed cleavage sites information Table S3. However, this issue can be resolved by using a sample tube with a volume that is preferably 10 times larger than that of the sample. Percentage of carbamylated peptides from serum proteins digested in different buffers with urea.
Human serum proteins were denatured, reduced, alkylated, and digested in different buffers 0. The percentage of peptides assigned to different forms of carbamylated peptides was determined based on spectral counts.
The possible mechanism of carbamylation inhibition by ammonium bicarbonate is as follows Fig. Therefore, the cyanate concentration should have decreased at least fold in the 1. Possible mechanism of carbamylation inhibition by ammonium containing buffer. After incubating angiotensin with 1. Samples were acidified to pH 2. The most abundant.
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These are numbered as spot number 1 to 4. Supernatants were collected and evaporated to dryness.
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