Table 1 Comparison of previously reported sample preparation methods for LC–MS analysis
Sample | Peptide Extraction Procedure | Advantages | Disadvantages | Ref | ||
|---|---|---|---|---|---|---|
Sample Pretreatment | Digestion | Composition of Extraction Buffer | ||||
Heated treated meat (boiling, autoclaving) | (a,b)Homogenizing(c) Acetone precipitation Air-drying pellet | (b,d)Digesting Drying Acidificating Desalting | 7 M urea, 2 M thiourea, 4% CHAPS, 50 mM DTT, 0.4% carrier ampholytes, protease inhibitor cocktail | The extraction process is quite simple and easy to do because cold conditions are provided by the ice cold buffer There is additional acetone precipitation so the protein obtained is purer The reducting and alkylating stages are shorter (total 50 min) | Overnight aceton precipitation is quite time consuming, not suitable for routine analysis applications The additional drying process before redissolving and after digesting is time consuming | Sarah et al. (2016) |
Heat treated meat and plant (waterbath heating, boiling, high temperature sterilizing, frying, baking) | (a)Pulverizing Cooling in an ice water bath(b) Homogenizing an in ice water bath environment(c) | (d)Digesting Acidificating Desalting | 7 M urea, 2 M thiourea, 50 mM Tris–HCl (pH 8) | Does not require additional liquid nitrogen. Hence, it is more cost-efficient. The extraction temperature is more stable due to ice water bath environment, thereby reducing protein degradation The digestion process is quite simple | There are two stages to homogenize the sample, meaning it is less concise Long reducting and alkylating stages (1.5 h) | Li et al. (2018) |
Heated treated meat (boiling, grilling) | (a)Grinding in liquid nitrogen(b,c) | (d)Filtrating and washing Digesting(c,b) | 8 M urea, 2 M thiourea, 4% CHAPS | Sample homogenization can be smoother, the whole preparation step is very simple and concise | There is a possibility that not all the proteins are perfectly extracted because homogenization with the buffer extraction is not clearly mentioned during extraction Double-stage centrifugation after digestion is less concise | Wang et al. (2018) |
Jellies, marshmallows, pastilles, pharmaceutical-grade gelatin capsules | Cutting Heating in deionized water(c) Overnight cold acetone extraction(c) Protein pellet air-drying | (b)Heating Filtering Digesting Stopping digestion | 40 mM ammonium bicarbonate containing 9% acetonitrile (pH 8.2) | The additional process with acetone extraction increases the purity of the extracted gelatin | The protein extraction steps are long and time-consuming, not suitable for routine analysis. | Jannat et al. (2020) |
In-house dairy products (yoghurt, cheese, ice cream) | Precipitation of other protein using mercury (II) nitrate solution Filtering Filtrate extraction using picric acid(c) Gelatin draining | (b)Sonicating(c) Filtering(b,d) Digesting Detergent removal Adding standard tryptic digest internal calibrant and buffer(c) | 50 mM ammonium bicarbonate | Proteins other than gelatin can be eliminated, increasing the purity of extracted gelatin Suitable for application in dairy products | The sample preparation stages are very complex and complicated, not suitable for routine analysis The cost is more expensive because there is the addition of reagents such as the use of detergents, reduction and alkylation are not really needed | Yilmaz et al. (2013) |
Gelatin used in preparing charcuterie meats, fruit snacks (Jello), Solaray empty gelatin capsules | (b)Sonicating Ethanol precipitation Vacuum centrifuge drying | (b)Sonicating Heating Digesting Stopping digestion(c) | 50 mM ammonium bicarbonate (pH 8) | Ethanol precipitation can increase assay sensitivity, the gelatin obtained is purer | The sample preparation stages are long, not suitable for routine analysis | Yang et al. (2018) |
Commercial powdered gelatin | (b)Sonicating | Digesting Filtering | Phosphate-buffered saline (PBS) | Rapid, simple procedure, cost-effective | Limited/no research as to whether it can be applied to more complex matrices The method is relatively new and needs to be validated for other complex matrix samples | Cai et al. (2021) |
Pure gelatin standard | (b)Filtering | Digesting | 1% ammonium bicarbonate (pH 8) | Easy and simple steps, suitable for routine analysis | Not clearly mentioned whether it is suitable for gelatin extracted from complex matrices | |
Gelatin-containing beverages | Shaking in thermostatic bath Adding formic acid and cold methanol/chloroform Cooling in agitation Cool incubating(c) Cold methanol precipitation(c) Protein pellet drying(b,c) | (d)Digesting | 50 mM ammonium bicarbonate | Multiple stages of protein precipitation — the protein obtained can be purer Shorter time for reduction and alkylation | Sample preparation stages are long and not suitable for routine analysis Reduction and alkylation are not quite necessary, using more enzymes for digestion | Dal Bello et al. (2021) |