Recent Advances in Methods for Measuring Plant Protein-Related Indices and Application-Oriented Selection
Recent Advances in Methods for Measuring Plant Protein-Related Indices and Application-Oriented Selection
Plant proteins are among the most important nitrogen-containing organic substances in plant tissues. They contribute to structural assembly and maintenance of metabolic enzyme systems, and they also serve as core indicators for quality evaluation and processing suitability. Different “protein-related indices” do not describe the same analytical target. Soluble protein more closely reflects cellular metabolic activity and the extractable protein pool; albumin, globulin, prolamin, and glutelin fractions represent solubility-based classes of storage proteins and their structural features; true protein and non-protein nitrogen distinguish protein-bound nitrogen from low-molecular-weight nitrogenous compounds; and gluten, acid detergent insoluble protein, and neutral detergent insoluble protein are closely associated with processing quality, cell-wall-associated proteins, or poorly soluble protein fractions. Selecting a method system aligned with the research objective, and standardizing extraction definitions, nitrogen determination schemes, blanks, and QC strategies, are essential for generating comparable data and robust conclusions.
Keywords: plant protein; soluble protein; protein fractions; true protein; non-protein nitrogen; TVBN; Kjeldahl nitrogen determination; Coomassie Brilliant Blue; gluten; detergent-insoluble protein
I. Quantitative Determination of Soluble Protein
1.1 Soluble Protein: Coomassie Brilliant Blue Colorimetric Assay
(1) Principle
Under acidic conditions, Coomassie Brilliant Blue G-250 binds to proteins (particularly at sites associated with basic and aromatic amino acid residues), inducing a dye conformational shift and a change in the absorption maximum. Protein concentration is quantified by spectrophotometric measurement of absorbance and comparison with a standard curve. This method is suitable for relative or absolute comparisons of “extractable/soluble proteins” in plant tissues, and it is widely used in stress physiology, growth and development studies, and assessments of metabolic status.
(2) Key Procedural Considerations
① Sampling and protein stability control: Standardize sampling tissue and time, and process samples rapidly under low temperature. For tissues with high protease activity, minimize exposure time to reduce degradation.
② Standardization of the extraction system: Define and fix the buffer system, solid-to-liquid ratio, extraction duration, and temperature. Extraction conditions determine the operational definition of “soluble protein” and should be consistent within a study.
③ Clarification and interference management: Centrifuge to remove insoluble material. For matrices rich in pigments, phenolics, or viscous polysaccharides, evaluate background contribution to optical measurements and perform clarification and/or background subtraction as needed.
④ Consistent color development: Standardize dye preparation, addition sequence, mixing, and the reaction time window to avoid within-batch drift caused by kinetic differences.
⑤ Standard curve and linear range verification: Prepare a concentration series using bovine serum albumin (BSA) standards and confirm the linear range. Dilute high-protein extracts to ensure measurements fall within the valid linear region.
⑥ Quality control: Include reagent blanks and sample blanks. A mid-level verification point or QC sample is recommended to monitor between-batch consistency.
(3) Advantages, Limitations, and Applicability
① Advantages: Relatively high sensitivity, operational convenience, and suitability for high-throughput measurements.
② Limitations: Response factors differ among proteins; certain surfactants, strong acids/bases, or high salt concentrations may affect binding and signal stability.
③ Use scenarios: Stress-response and physiological-status evaluation; trend analysis of extractable protein pool dynamics; batch comparisons across multiple treatments or time points.
II. Solubility-Based Fractionation and Quantification of Protein Components
2.1 Protein Fractions: Albumin/Globulin/Prolamin/Glutelin (Sequential Extraction with Continuous Agitation)
(1) Principle
Based on differences in protein solubility, sequential extraction is performed using a defined series of solvent systems: predominantly water-soluble albumins, salt-soluble globulins, alcohol-soluble prolamins, and alkali-soluble glutelins. Fractionated extracts enable estimation of each component, supporting studies on storage protein composition, quality classification, and cultivar/line differences.
(2) Key Procedural Considerations
① Sample pretreatment and homogenization: Thoroughly grind and homogenize samples to reduce fraction bias caused by tissue heterogeneity. For lipid-rich materials, optional defatting may improve fractionation consistency.
② Fixed extraction order and conditions: Do not alter the extraction sequence. Keep solvent concentration, solid-to-liquid ratio, agitation intensity, extraction time, and temperature strictly consistent to maintain a stable operational definition of each “fraction.”
③ Inter-step separation and residue handling: After each step, separate supernatant by centrifugation. Remove residual solvent from the pellet as much as possible before the next step to reduce carryover and cross-contamination.
④ Consistent quantification strategy: Quantify each extract using a unified protein assay (e.g., colorimetry) or a predefined nitrogen-based conversion scheme. Maintain the same reporting convention within a study.
⑤ Reproducibility and recovery checks: Include replicates. When necessary, evaluate systematic bias by checking agreement between total protein and the sum of fractions (within expected method error).
(3) Advantages, Limitations, and Applicability
① Advantages: Provides compositional information, facilitating interpretation and association analyses of storage protein differences.
② Limitations: Sensitive to extraction conditions; inter-fraction carryover can shift apparent composition; solubility overlap exists, so results represent operational rather than absolute biochemical classes.
③ Use scenarios: Grain quality and processing suitability research; compositional shifts induced by genotype or treatment.
III. Nitrogen-Related Indices and Discrimination of Protein-Bound vs. Non-Protein Nitrogen
3.1 Total Volatile Basic Nitrogen (TVBN): Semi-Micro Kjeldahl Method
(1) Principle
TVBN reflects the content of volatile basic nitrogenous compounds and is commonly used to evaluate accumulation of low-molecular-weight nitrogen species and associated nitrogen-metabolism features linked to quality changes. The semi-micro Kjeldahl system quantifies nitrogen through digestion, distillation/absorption, and titration.
(2) Key Procedural Considerations
① Sample handling and volatilization control: Prepare samples to minimize losses of volatile components; avoid prolonged exposure to high temperature or strong alkali during handling.
② Unified digestion system and catalytic conditions: Fix reagent composition, digestion temperature, and time to ensure complete conversion and between-batch consistency.
③ Standardized distillation, absorption, and titration: Keep absorber concentration, distillation time, and endpoint criteria consistent. Regularly standardize titrant solutions to ensure accuracy.
④ Blanks and QC: Include reagent blanks and subtract accordingly; introduce QC samples to monitor intra-day and inter-day stability.
(3) Advantages, Limitations, and Applicability
① Advantages: Strong traceability and suitability for nitrogen-based quantitative comparisons.
② Limitations: Time-consuming; requires strict operational consistency and titration proficiency.
③ Use scenarios: Evaluation of volatile nitrogen accumulation; monitoring indices related to quality changes or nitrogen metabolism.
3.2 True Protein: Trichloroacetic Acid (TCA) Precipitation Coupled with Kjeldahl Nitrogen Determination
(1) Principle
TCA precipitates proteinaceous, high-molecular-weight nitrogen-containing compounds while retaining non-protein small-molecule nitrogen in the supernatant. Kjeldahl determination on the precipitate yields the protein-bound nitrogen level corresponding to “true protein.”
(2) Key Procedural Considerations
① Fixed precipitation conditions: Standardize TCA concentration, addition ratio, precipitation duration, and temperature to ensure stable precipitation efficiency.
② Pellet washing and transfer: Washing reduces carryover of supernatant non-protein nitrogen that would otherwise cause positive bias; minimize mechanical loss during transfer to avoid underestimation.
③ Consistent digestion and titration workflow: Follow standardized Kjeldahl procedures for digestion, distillation, and titration, with blanks and QC samples controlling batch variability.
④ Harmonized reporting: Report true protein as nitrogen content or as protein equivalents using a defined N-to-protein conversion factor; keep the convention consistent within the study.
(3) Advantages, Limitations, and Applicability
① Advantages: Separates protein-bound nitrogen from non-protein nitrogen, improving interpretability relative to total nitrogen alone.
② Limitations: Accuracy depends on precipitation efficiency and washing adequacy; workflow is relatively labor-intensive.
③ Use scenarios: Partitioning protein-bound vs. non-protein nitrogen; assessing protein degradation trends or non-protein nitrogen accumulation.
3.3 Non-Protein Nitrogen (NPN): Sodium Tungstate Precipitation or TCA Precipitation
(1) Principle
Proteinaceous macromolecular nitrogen is removed by precipitation, while low-molecular-weight nitrogenous compounds (amino acids, amines, small peptides, etc.) remain in the clarified supernatant. Nitrogen (or a defined analytical signal) in the supernatant is quantified to obtain NPN levels.
(2) Key Procedural Considerations
① Precipitant selection and unified conditions: Sodium tungstate and TCA differ in selectivity; fix the precipitant and operating conditions within a study.
② Adequate clarification: Ensure complete clarification of the supernatant after precipitation; fine particulate carryover can reduce reproducibility and bias NPN estimates.
③ Matched quantification convention: Quantify supernatant nitrogen using an established nitrogen determination scheme, ideally paired with the true-protein workflow to enable a closed mass-balance framework (“protein-bound N + NPN”).
④ Quality control: Include blanks and replicates; assess repeatability and recovery to verify stability of the separation step.
(3) Advantages, Limitations, and Applicability
① Advantages: Captures accumulation of low-molecular-weight nitrogen species, often closely linked to protein degradation and nitrogen remobilization.
② Limitations: Selectivity differs across precipitation systems; results are not directly interchangeable across methods; complex matrices are vulnerable to incomplete precipitation.
③ Use scenarios: Monitoring protein degradation or NPN accumulation; combined interpretation with true protein for nitrogen partitioning.
IV. Indices Associated with Processing Quality or Poorly Soluble Protein Fractions
4.1 Gluten: Hand-Washing Method
(1) Principle
During washing of dough with water, starch and soluble components are removed, leaving a residue mainly composed of the gluten protein network. Gluten content is obtained by weighing and/or calculation according to a standardized procedure, reflecting gluten network-forming capacity and processing-related quality traits.
(2) Key Procedural Considerations
① Consistent dough formation: Water addition, kneading time, and resting conditions affect gluten network development and should follow a fixed protocol.
② Standardized washing procedure: Keep washing intensity, duration, and water-change frequency consistent to avoid under-washing or over-washing.
③ Unified endpoint determination: Define the endpoint using extract clarity or specified criteria to minimize operator subjectivity.
④ Reporting and repeatability: Include replicates and report results using a consistent convention (e.g., wet gluten vs. dry gluten).
(3) Advantages, Limitations, and Applicability
① Advantages: Directly linked to processing suitability for dough-based products; strong process interpretability.
② Limitations: Operator-dependent; reproducibility relies on strict procedural control and proficiency.
③ Use scenarios: Processing quality evaluation for wheat and related cereals; cultivar selection and quality grading.
4.2 Acid Detergent Insoluble Protein (ADIP): Acid Detergent Extraction Coupled with Kjeldahl Nitrogen Determination
(1) Principle
An acid detergent washing system is used to extract and separate nitrogenous components associated with cell walls/fiber fractions or poorly soluble proteins. Kjeldahl nitrogen determination on the residue or target fraction provides an ADIP-related nitrogen level.
(2) Key Procedural Considerations
① Fixed detergent system and conditions: Standardize detergent formulation, temperature, duration, and mixing intensity to maintain a stable separation boundary.
② Residue recovery and adequate washing: Avoid residue loss; washing adequacy affects residual soluble nitrogen carryover and thus quantification accuracy.
③ Standardized Kjeldahl workflow: Execute digestion, distillation, titration, and blank correction rigorously.
④ Structure-linked interpretation: This index often correlates with fiber/cell-wall fractions and poorly soluble protein proportion; interpret jointly with relevant structural indices.
(3) Advantages, Limitations, and Applicability
① Advantages: Characterizes poorly soluble or bound nitrogen fractions, supporting structure–quality association studies.
② Limitations: Sensitive to washing/separation conditions; workflow is relatively labor-intensive.
③ Use scenarios: Comparative evaluation of cell-wall-associated/insoluble protein fractions; structural assessments relevant to feed or processing quality.
4.3 Neutral Detergent Insoluble Protein (NDIP): Neutral Detergent Extraction Coupled with Kjeldahl Nitrogen Determination
(1) Principle
A neutral detergent system separates cell-wall-related components while retaining specific insoluble nitrogen fractions. Kjeldahl determination on the resulting fraction yields an NDIP-related nitrogen level, often used to evaluate nitrogen associated with structural carbohydrates.
(2) Key Procedural Considerations
① Consistent neutral detergent conditions: Fix formulation, processing temperature, and duration to reduce between-batch variability.
② Stable separation and recovery: Standardize centrifugation/filtration conditions to avoid bias from fine particle carryover.
③ Kjeldahl QC: Ensure traceability via blank subtraction, titrant standardization, and QC sample monitoring.
④ Explicit interpretive boundary: This index is an operational fraction reflecting detergent-insoluble nitrogen; it is best suited for relative comparisons and trend analyses under strictly matched conditions.
(3) Advantages, Limitations, and Applicability
① Advantages: Useful for characterizing structurally bound nitrogen or poorly soluble protein fractions; facilitates integrated interpretation with fiber/cell-wall indices.
② Limitations: Results are condition-sensitive; comparability across studies depends on strictly matched detergent protocols.
③ Use scenarios: Evaluation of insoluble nitrogen fractions; feed-quality or structure-related quality research.
V. Overall Comparison of Indices and Methods and Application-Oriented Selection
Index | Recommended method | Sensitivity | Key control points and common interferences | Use scenarios |
Soluble protein | Coomassie Brilliant Blue assay | Medium–High | Extraction definition, reaction time window, background absorbance; interference from pigments/phenolics/surfactants | Physiological status assessment; stress response; batch comparisons |
Protein fractions (Alb/Glo/Pro/Glu) | Sequential extraction with agitation | Medium | Extraction order/conditions; inter-step carryover; solubility overlap | Storage protein profiling; quality typing; cultivar differences |
TVBN | Semi-micro Kjeldahl | Medium | Consistency of digestion/distillation/titration; volatile losses; endpoint judgment | Volatile nitrogen evaluation; monitoring quality-change-related traits |
True protein | TCA precipitation + Kjeldahl | Medium | Precipitation efficiency and washing; pellet transfer loss; titration standardization | Protein-bound nitrogen assessment; protein degradation trend |
Non-protein nitrogen | Sodium tungstate or TCA precipitation | Medium | Selectivity differences; supernatant clarity; non-interchangeable conventions | Low-molecular-weight nitrogen accumulation; paired with true protein |
Gluten | Hand-washing method | Medium | Dough formation; washing intensity and endpoint; operator consistency | Processing suitability; cultivar screening |
ADIP | Acid detergent extraction + Kjeldahl | Medium | Washing conditions and residue recovery; residual soluble N; titration consistency | Insoluble/bound N assessment; structure association |
NDIP | Neutral detergent extraction + Kjeldahl | Medium | Washing conditions; separation/recovery; particle carryover; titration consistency | Structurally bound nitrogen; feed-quality-related research |
VI. Method Combinations and Experimental Design Recommendations
6.1 Studies on Physiological Status and Stress Responses
(1) Recommended combination: soluble protein (Coomassie assay) + non-protein nitrogen (precipitation-based separation) or true protein (TCA precipitation + Kjeldahl).
(2) Interpretive framework: Soluble protein reflects changes in the extractable protein pool, while true protein/NPN discriminates protein-bound nitrogen from small-molecule nitrogen redistribution, strengthening inference on degradation vs. accumulation processes.
6.2 Quality Typing and Processing Suitability Evaluation
(1) Recommended combination: fractionation of protein components (Alb/Glo/Pro/Glu) + gluten (hand-washing).
(2) Key requirements: Strictly standardize fractionation order and conditions; for gluten, standardize dough formation and washing endpoint determination to minimize operator variability.
6.3 Evaluation of Structurally Bound Proteins and Insoluble Nitrogen Fractions
(1) Recommended combination: ADIP + NDIP, benchmarked against true protein or total nitrogen.
(2) Key requirements: Keep detergent formulation, temperature/time, and separation/recovery procedures consistent; use blanks and QC samples to monitor between-batch comparability.
VII. Aladdin-Related Products
Name | CAS No. | Applicable indices | Typical use/notes |
Coomassie Brilliant Blue G-250 | Soluble protein (Coomassie Brilliant Blue colorimetric assay) | Dye-binding color development | |
Phosphoric acid | Soluble protein (Coomassie Brilliant Blue colorimetric assay) | pH adjustment to maintain acidic color-developing conditions | |
Ethanol | Soluble protein (Coomassie Brilliant Blue colorimetric assay); protein fractions (prolamin extraction) | Solvent for dye system; alcohol-based fractionation extraction | |
Bovine serum albumin (BSA) | Soluble protein (Coomassie Brilliant Blue colorimetric assay) | Standard for calibration curve | |
Sodium chloride | Protein fractions (globulin extraction) | Salt-soluble fractionation extraction | |
Sodium hydroxide | Protein fractions (glutelin extraction); TVBN/Kjeldahl system (alkalization for distillation) | Alkali extraction; alkalization prior to distillation | |
Sulfuric acid | TVBN (Kjeldahl nitrogen determination); true protein (precipitation–Kjeldahl); acid/neutral detergent insoluble protein (Kjeldahl) | Acid for Kjeldahl digestion | |
Potassium sulfate | TVBN (Kjeldahl nitrogen determination); true protein (precipitation–Kjeldahl); acid/neutral detergent insoluble protein (Kjeldahl) | Digestion boiling-point elevating agent | |
Copper(II) sulfate | TVBN (Kjeldahl nitrogen determination); true protein (precipitation–Kjeldahl); acid/neutral detergent insoluble protein (Kjeldahl) | Digestion catalyst | |
Boric acid | TVBN (Kjeldahl nitrogen determination); true protein (precipitation–Kjeldahl); acid/neutral detergent insoluble protein (Kjeldahl) | Distillate absorbing solution | |
Hydrochloric acid standard solution | TVBN (Kjeldahl nitrogen determination); true protein (precipitation–Kjeldahl); acid/neutral detergent insoluble protein (Kjeldahl) | Standard solution for titration | |
Trichloroacetic acid (TCA) | True protein (TCA precipitation–Kjeldahl); non-protein nitrogen (TCA precipitation) | Protein precipitation and separation | |
Sodium tungstate | Non-protein nitrogen (sodium tungstate precipitation) | Protein precipitation and separation |
Plant protein-related indices provide a multilayered framework, spanning the extractable soluble protein pool, storage protein composition, partitioning between protein-bound and non-protein nitrogen, and processing-quality-relevant as well as detergent-insoluble/bound nitrogen fractions. The Coomassie assay enables high-throughput profiling of soluble protein dynamics. Sequential fractionation provides compositional resolution of storage proteins. Kjeldahl-based systems, coupled with precipitation or detergent-washing separations, support traceable quantification and discrimination among nitrogen forms. Gluten determination directly serves processing suitability evaluation. In practice, index selection should be driven by the research question, and comparability and reproducibility should be ensured through standardized extraction definitions, digestion–distillation–titration workflows, and rigorous blank and QC strategies, thereby strengthening scientific inference and the generalizability of conclusions.
For more related articles, please see below:
[1] Research Progress in Detection Technologies for Soluble Sugars in Plants
[2] Aladdin® Plant Research Related Products
[3] Suitable for Plant Cell Culture
[5] Experiments on bidirectional electrophoresis techniques in plant proteomics