This article describes a sample preparation method based on heat-inactivation to preserve endogenous peptides avoiding degradation post-mortem, followed by relative quantitation using isotopic labeling plus LC-MS.
Peptidomics can be defined as the qualitative and quantitative analysis of peptides in a biological sample. Its main applications include identifying the peptide biomarkers of disease or environmental stress, identifying neuropeptides, hormones, and bioactive intracellular peptides, discovering antimicrobial and nutraceutical peptides from protein hydrolysates, and can be used in studies to understand the proteolytic processes. The recent advance in sample preparation, separation methods, mass spectrometry techniques, and computational tools related to protein sequencing has contributed to the increase of the identified peptides number and peptidomes characterized. Peptidomic studies frequently analyze peptides that are naturally generated in cells. Here, a sample preparation protocol based on heat-inactivation is described, which eliminates protease activity, and extraction with mild conditions, so there is no peptide bonds cleavage. In addition, the relative quantitation of peptides using stable isotope labeling by reductive methylation of amines is also shown. This labeling method has some advantages as the reagents are commercially available, inexpensive compared to others, chemically stable, and allows the analysis of up to five samples in a single LC-MS run.
"Omics" sciences are characterized by the deep analysis of a molecule set, such as DNA, RNA, proteins, peptides, metabolites, etc. These generated large-scale datasets (genomics, transcriptomics, proteomics, peptidomics, metabolomics, etc.) have revolutionized biology and led to an advanced understanding of biological processes1. The term peptidomics began to be introduced in the early 20th century, and some authors have referred to it as a branch of proteomics2. However, peptidomics has distinct particularities, where the main interest is to investigate the naturally generated peptides content during cellular processes, as well as the characterization of biological activity of these molecules3,4.
Initially, bioactive peptide studies were restricted to the neuropeptides and hormone peptides through Edman degradation and radioimmunoassay. However, these techniques do not allow a global analysis, depending on the isolation of each peptide in high concentrations, time for the generation of antibodies, besides cross-reactivity possibility5.
Peptidomics analysis was only made possible after several advances in Liquid chromatography coupled mass spectrometry (LC-MS) and genome projects that delivered comprehensive data pools for proteomics/peptidomics studies6,7. Moreover, a specific peptide extraction protocol for peptidomes needed to be established because the first studies that analyzed neuropeptides globally in brain samples showed that detection was affected by the massive degradation of proteins, which occur mainly in this tissue after 1 min post-mortem. The presence of these peptide fragments masked the neuropeptide signal and did not represent the peptidome in vivo. This problem was solved mainly with the application of fast heating inactivation of proteases using microwave irradiation, which drastically reduced the presence of these artifact fragments and allowed not only the identification of neuropeptide fragments but revealed the presence of a set of peptides from cytosolic, mitochondrial, and nuclear proteins, different of degradome6,8,9.
These methodological procedures allowed an expansion of the peptidome beyond the well-known neuropeptides, where hundreds of intracellular peptides generated mainly by the action of proteasomes have been identified in yeast10, zebrafish11, rodent tissues12, and human cells13. Dozens of these intracellular peptides have been extensively shown to have both biological and pharmacological activities14,15. Furthermore, these peptides can be used as disease biomarkers and possibly have clinical significance, as demonstrated in cerebrospinal fluid from patients with intracranial saccular aneurysms16.
Currently, in addition to the identification of peptide sequences, it is possible through mass spectrometry to obtain data of absolute and relative quantitation. In the absolute quantitation, the peptide levels in a biological sample are compared to synthetic standards, while in the relative quantitation, the peptide levels are compared among two or more samples17. Relative quantitation can be performed using the following approaches: 1) "label free"18; 2) in vivo metabolic labeling or 3) chemical labeling. The last two are based on the use of stable isotopic forms incorporated into peptides19,20. In label-free analysis, the peptide levels are estimated by considering the signal strength (spectral counts) during the LC-MS18. However, isotopic labeling can obtain more accurate relative levels of peptides.
Many peptidomic studies used trimethylammonium butyrate (TMAB) labeling reagents as chemical labeling, and more recently, Reductive Methylation of Amines (RMA) with deuterated and non-deuterated forms of formaldehyde and sodium cyanoborohydride reagents have been used11,21,22. However, the TMAB labels are not commercially available, and the synthesis process is very laborious. On the other hand, in the RMA, the reagents are commercially available, inexpensive compared to other labels, the procedure is simple to perform, and the labeled peptides are stable23,24.
The use of RMA involves forming a Schiff base by allowing the peptides to react with formaldehyde, followed by a reduction reaction through the cyanoborohydride. This reaction causes dimethylation of free amino groups on N-terminals and lysine side chains and monomethylates N-terminal prolines. How proline residues are often rare on the N-terminal, practically all peptides with free amines on the N-terminus are labeled with two methyl groups23,24,25.
The following procedure for peptide extraction and reductive methylation was adapted from previously published procedures24,25,26,27. This protocol followed the guidelines of the National Council for Animal Experimentation Control (CONCEA) and was approved by the Ethics Commission for Animal Use (CEUA) at Bioscience Institute of Sao Paulo State University. The protocol steps are shown in Figure 1.
NOTE: Prepare all aqueous solutions in ultrapure water.
1. Peptide extraction
2. Peptide quantification with fluorescamine
NOTE: The amount of peptide can be estimated using fluorescamine at pH 6.8 as previously described11,28. This method consists of the attachment of a fluorescamine molecule to the primary amines present in the lysine (K) residues and/or the N-terminal of peptides. The reaction is performed at pH 6.8 to guarantee that the fluorescamine reacts only with the amino groups of the peptides and not with free amino acids. The fluorescamine is measured by using a spectrofluorometer at an excitation wavelength of 370 nm and an emission wavelength of 480 nm.
3. Reductive methylation of amines labeling
NOTE: This isotopic labeling method is based on the dimethylation of amine groups with deuterated and non-deuterated forms of formaldehyde and sodium cyanoborohydride reagents. The final product of this reaction adds 28 Da, 30 Da, 32 Da, 34 Da, or 36 Da to the final mass of each peptide at each available labeling site (lysine or N-terminal). This reaction produces an m/z difference in the peptides labeled with different forms observed in the MS spectrum (Table 1).
CAUTION: Proper safety equipment should be used to handle these compounds, and care should be taken to minimize exposure. Procedures with formaldehyde and sodium cyanoborohydride reagents should be performed in a fume hood because they are very toxic (including weighing the sodium cyanoborohydride). During the quenching reaction and acidification, a toxic gas (hydrogen cyanide) may be generated.
4. Liquid chromatography and mass spectrometry
5. Relative quantitation of peptides
NOTE: The MS spectra are analyzed in the mass spectrometer software. Peak groups of labeled peptides with different tags are identified in the MS spectra. The relative quantitation is calculated by the intensity of each monoisotopic peak. Each treated group is compared to the respective control group.
6. Peptide Identification
Figure 1: Peptidomic studies workflow. Steps of peptide extraction and Reductive Methylation of amines. Please click here to view a larger version of this figure.
The results obtained from the runs carried out on the mass spectrometer are stored in raw data files that can be opened in the mass spectrometer software. In the MS spectra, it is possible to observe peak groups representing labeled peptides according to the labeling scheme used, ranging from 2-5 labels. For example, in Figure 2, pairs of peaks detected in a chromatographic time are represented in an experiment where only two isotopic labels were used in two different samples in the same run. Figure 3 shows other possibilities of positive results, using 3 and 4 different labels in each LC-MS run. When using 4 or 5 labels in an LC/MS run, there may be an overlap of peaks of the labeled peptides with the different tags that need to be corrected to obtain the real intensity value of each peak (Figure 4).
The isotopic labeling can also be used to show substrates and products in vitro for a given protease or peptidase, as shown in Figure 5. Finally, different software can be used to identify the labeled peptides, such as Peaks Studio or MASCOT. These software applications were created for proteomic analysis; therefore, the protein quantitation data should not be considered for peptidomics analysis, and each labeled peptide identified within the reliability parameters must be checked and then quantified. If the peptides have been successfully detected and labeled, these programs will provide a list of identified peptide sequences containing the labels. Figure 6 is shown an example of the identification of a peptide sequence done by the program. In this case, only 3 different forms of the labels (L1, L3, and L5) were used to label three different samples separately, which were then mixed and analyzed by mass spectrometry in a single run.
Figure 2: MS spectrum representative of a chromatographic time accumulated in a typical labeling experiment using reductive dimethylation of amines. In (A), the red arrows indicate the presence of peak pairs of different peptides labeled with 2 isotopic forms (L1 and L5) for comparison between two different samples (S1 and S2). In (B), an enlarged image of an MS spectrum of the same peptide showing different m/z because of the use of labels. In this case, there was no variation in the peak intensity for this peptide present in these samples. Please click here to view a larger version of this figure.
Figure 3: Representative MS spectrum of labeled peptides with reductive methylation of amines using different numbers of tags. (A) A triplex labeling was used. It is possible to observe an MS spectrum of a peptide present in 3 different samples (S1, S2, and S3) labeled with L1, L3, and L5 tags, respectively. In this case, the level of the labeled peptide with L5 was twice the level observed for the same peptide labeled with L1 and L3 tags. (B) A quadripex labeling was performed using the L1, L2, L3, and L4 labels. In this case, control samples (S1 and S3) were labeled with L1 and L3, respectively, and compared with two experimental samples (S2 and S4) labeled with L2 and L4 labels. No significant differences were observed for this peptide between the samples. Please click here to view a larger version of this figure.
Figure 4: Representative MS spectrum of a labeled peptide presenting a peak overlap. This figure shows the MS spectrum of a peptide of charge 3, mass of 2098.87 Da with a single primary amine available for labeling. The difference between the labeled peptides is only 2 Da, causing an overlap when 4 or 5 labels are used in the same LC/MS run. Using a model based on cubic polynomial equations, it is possible to correct these overlaps between the labels. In the graph, the red bars show the average of the intensity values adjusted for this peptide in two runs. The black bars show the average of the overlapping intensity values. After correction, this peptide showed little variation in intensity between samples (red bars). Please click here to view a larger version of this figure.
Figure 5: Representative MS spectrum of labeled peptides with reductive methylation of amines in a proteolysis study. Here, peptide extracts were incubated with 200 nM and 20 nM neurolysin to characterize their substrates and products. For confirmation of the result, two LC/MS runs with Forward and Reverse labeling strategies were performed. In the second run the position of the samples is changed in the labeling procedure in relation to the scheme used in the first run. In A, it is shown a peptide that does not change (NC) in the presence of the neurolysin enzyme. In B, a peptide that disappeared in the presence of a high concentration of the enzyme (S2) and that showed a small reduction in the low concentration of the enzyme (S4) in both forward and reverse labeling. This peptide is considered a substrate (SB) of the enzyme. In C an example of a peptide that was considered a product (PD), because its concentration was increased (S2 and S4) in the presence of the enzyme. Please click here to view a larger version of this figure.
Figure 6: Representative MSMS spectrum and identification of a labeled peptide performed by a database search engine. In this example, it is possible to observe 2+ ions with m/z 672,3802, 676,4045, and 680,4241 corresponding to a peptide of the same mass labeled with the L1, L3, and L5 methylated forms, respectively. This sequence ADQVSASLAKQGL was identified through the MS/MS spectrum as a fragment of the microtubule-associated protein tau isoform X1. This peptide has the N-terminal and a lysine available as labeling sites adding a mass difference of 8 Daltons between the labeled peptides. The MS spectrum of this peptide is shown in Figure 3A. Please click here to view a larger version of this figure.
Sample | H2CO | D2CO | D213CO | NaBH3CN | NaBD3CN | Label | Additional Mass |
2 x 4 µL | 2 x 4 µL | 2 x 4 µL | 2 x 4 µL | 2 x 4 µL | code | (Da) | |
1 | X | X | L1 | 28.0313 | |||
2 | X | X | L2 | 30.0439 | |||
3 | X | X | L3 | 32.0564 | |||
4 | X | X | L4 | 34.069 | |||
5 | X | X | L5 | 36.0757 |
Table 1: Reagents of a typical experiment using the indicated combination of deuterated and non-deuterated forms of formaldehyde and sodium cyanoborohydride.
In most peptidomics studies, one of the critical steps is, without doubt, the sample preparation that should be carefully performed to avoid the presence of peptide fragments generated by proteases after a few minutes post-mortem. The initial studies on brain extracts prepared from non-microwaved samples showed a large number of protein fragments to be present in the 10-kDa microfiltrates. Different approaches have been described to avoid peptide spectra from protein degradation: focused microwave irradiation animal sacrifice6,8, cryostat dissection followed by a boiling extraction buffer31, and post-sacrifice microwave-irradiation of tissue using a household type microwave oven9,26. For cell culture and some tissues, the protease inactivation can be done directly by adding water at 80 °C. However, some samples, such as nervous tissue, can be more sensitive to post-mortem change, and protease inactivation by microwave irradiation has been indicated as a choice method. Furthermore, another important point during the peptide extraction is to make sure that extracts are ice-cold before adding acid to prevent acid-labile bonds from breaking, like cleavage of Asp-Pro bonds26.
Different strategies can be used for the relative quantification of peptides, but none of them can be considered completely ideal. To choose the method to be used, the researcher must consider factors such as availability of hours of use in the mass spectrometer, commercially available and costs of labeling reagents, and ease in analyzing the data obtained17,25,26,32. The label-free method has been widely used but requires many hours in the mass spectrometer. It is necessary to inject technical replicates for each sample and depends on chromatographic reproducibility among the samples. Other chemical labeling reagents, for example, ITRAQ (isobaric tags for relative and absolute quantitation) and TMT (tandem mass tag), are expensive and only provide quantitation of peptides selected for MS/MS analysis25,33,34.
The major limitation of relative quantification through chemical labeling using RMA is the overlap that occurs for some peptides when performing a protocol using 4 or 5 labels at the same run resulting in mass differences of 2 Da for peptides with a single primary amine and 1 Da for peptides with N-terminus proline and no internal lysine residues. However, Tashima and Fricker (2018) developed a model to correct isotopic overlapping based on cubic polynomial equations25, which get the correct intensity of the labeled peptides in the samples. Furthermore, not all peptides can be seen by RMA. For example, some peptides lack an N-terminal free amine due to acetylation, pyroglutamylation, or another modification. If internal lysines are also absent in these peptides, they will not be labeled by the RMA reagent and will appear on the m/z spectra as unquantifiable single peaks27.
The authors have nothing to disclose.
The development and use of the techniques described here were supported by the Brazilian National Research Council grant 420811/2018-4 (LMC); Fundação de Amparo à Pesquisa do Estado de São Paulo (www.fapesp.br) grants 2019/16023-6 (LMC), 2019/17433-3 (LOF) and 21/01286-1 (MEME). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the article.
10 kDa cut-off filters | Merck Millipore | UFC801024 | Amicon Ultra-4, PLGC Ultracel-PL Membrane, 10 kDa |
Acetone | Sigma-Aldrich | 179124 | |
Acetonitrile | Sigma-Aldrich | 1000291000 | |
Ammonium bicarbonate | Sigma-Aldrich | 11213 | |
analytical column (EASY-Column) | EASY-Column | (SC200) | 10 cm, ID75 µm, 3 µm, C18-A2 |
Ethyl 3-aminobenzoate methanesulfonate | Sigma-Aldrich | E10521 | MS-222 |
Fluorescamine | Sigma-Aldrich | F9015 | |
Formaldehyde solution | Sigma-Aldrich | 252549 | |
Formaldehyde-13C, d2, solution | Sigma-Aldrich | 596388 | |
Formaldehyde-d2 solution | Sigma-Aldrich | 492620 | |
Formic acid | Sigma-Aldrich | 33015 | |
Fume hood | Quimis | Q216 | |
Hydrochloric acid – HCl | Sigma-Aldrich | 258148 | |
LoBind-Protein retention tubes | Eppendorf | EP0030108116-100EA | |
LTQ-Orbitrap Velos | Thermo Fisher Scientific | LTQ Velos | |
Microwave oven | Panasonic | NN-ST67HSRU | |
n Easy-nLC II nanoHPLC | Thermo Fisher Scientific | LC140 | |
PEAKS Studio | Bioinformatics Solutions Inc. | VERSION 8.5 | |
Phosphate-buffered saline | Invitrogen | 3002 | tablets |
precolumn (EASY-Column) | Thermo Fisher Scientific | (SC001) | 2 cm, ID100 µm, 5 µm, C18-A1 |
Refrigerated centrifuge | Hermle | Z326K | for conical tubes |
Refrigerated centrifuge | Vision | VS15000CFNII | for microtubes |
Reversed-phase cleanup columns (Oasis HLB 1 cc Cartridge) | Waters | 186000383 | Oasis HLB 1 cc Cartridge |
Sodium cyanoborodeuteride – NaBD3CN | Sigma-Aldrich | 190020 | |
Sodium cyanoborohydride – NaBH3CN | Sigma-Aldrich | 156159 | |
Sodium phosphate dibasic | Sigma-Aldrich | S9763 | NOTE: 0.2 M PB= 0.1 M phosphate buffer pH 6.8 (26.85 mL of Na2HPO3 1M) plus 0.1 M phosphate buffer pH 6.8 (23.15 mL of NaH2PO3 1M) to 250 ml of water |
Sodium phosphate monobasic | Sigma-Aldrich | S3139 | |
Sonicator | Qsonica | Q55-110 | |
Standard peptide | Proteimax | amino acid sequence: LTLRTKL | |
Triethylammonium buffer – TEAB 1 M | Sigma-Aldrich | T7408 | |
Trifluoroacetic acid – TFA | Sigma-Aldrich | T6508 | |
Ultra purified water | Milli-Q | Direct-Q 3UV | |
Vacuum centrifuge | GeneVac | MiVac DNA concentrator | |
Water bath | Cientec | 266 | |
Xcalibur Software | ThermoFisher Scientific | OPTON-30965 |