Understanding Mass Spectrometry Results for Peptides

Mass spectrometry stands as the definitive analytical method for confirming peptide identity and purity. A Certificate of Analysis without valid MS data is essentially worthless. Here's what you need to know to interpret MS results correctly and spot fabricated data.

What Mass Spectrometry Measures

Mass spectrometry measures the mass-to-charge ratio (m/z) of ionized molecules. For peptides, this reveals the molecular weight with extreme precision, typically within 0.01-0.05% accuracy.

The process works through four fundamental steps:

Ionization: The peptide sample is converted into gas-phase ions. For peptides, electrospray ionization (ESI) is the standard method. ESI gently ionizes peptides by adding protons (H+) without fragmenting the molecule. This is critical because peptides are large, fragile molecules that would break apart under harsher ionization methods.

Acceleration: Ions are accelerated through an electric field. Their velocity depends on their mass-to-charge ratio. Lighter ions or ions with more charges move faster than heavier ions or ions with fewer charges.

Separation: Ions are separated based on their m/z ratio. Different analyzer types accomplish this differently. Time-of-flight (TOF) analyzers measure how long ions take to reach the detector. Quadrupole analyzers use oscillating electric fields to filter ions. Orbitrap analyzers trap ions in an electric field and measure their oscillation frequency.

Detection: A detector counts ions arriving at specific m/z values. The intensity at each m/z point indicates how many ions of that mass are present. More intense peaks mean higher abundance of that particular ion.

The output is a spectrum showing intensity on the y-axis and m/z on the x-axis. Each peak represents ions with a specific mass-to-charge ratio.

How to Read MS Results

A legitimate peptide MS spectrum contains specific features that confirm identity and reveal purity issues.

Understanding Charge States

Peptides typically carry multiple positive charges during ESI ionization. A peptide with molecular weight 1000 Da might appear at m/z 501 if it carries two charges ([M+2H]2+), or at m/z 334 if it carries three charges ([M+3H]3+).

The number of charges depends on the number of basic amino acids (lysine, arginine, histidine) and the N-terminus. Longer peptides generally carry more charges. A 10-residue peptide might show +2 and +3 charge states. A 30-residue peptide might show +3, +4, and +5 charge states.

To calculate molecular weight from a charge state peak:

Molecular Weight = (m/z × charge) - (charge × 1.008)

The 1.008 value represents the mass of the added protons. For a peak at m/z 501 with +2 charge: MW = (501 × 2) - (2 × 1.008) = 1002 - 2.016 = 999.98 Da.

All charge states should calculate to the same molecular weight. If they don't, something is wrong with the data.

Peak Patterns and Isotope Distribution

Natural isotopes create characteristic patterns. Carbon-13 occurs at 1.1% natural abundance. A peptide containing 50 carbon atoms will show a second peak (M+1) at approximately 55% the intensity of the main peak.

The isotope pattern provides two critical checks. First, the spacing between isotope peaks confirms charge state. Peaks separated by 1.0 m/z indicate +1 charge. Peaks separated by 0.5 m/z indicate +2 charge. Peaks separated by 0.33 m/z indicate +3 charge.

Second, the isotope pattern intensity ratio confirms molecular size. Small molecules (under 500 Da) show weak M+1 peaks. Medium peptides (1000-2000 Da) show M+1 peaks at 30-60% of M intensity. Large peptides (over 3000 Da) often show M+1 or M+2 peaks larger than the M peak.

Vendors sometimes provide "monoisotopic mass" calculated from the lowest-mass isotope peak. This is the correct theoretical mass for comparison.

Base Peak and Relative Intensity

The base peak (100% intensity) represents the most abundant ion. Other peaks are scaled relative to this. Different charge states will have different intensities based on ionization efficiency. The +2 charge state often dominates for peptides under 2000 Da. The +3 or +4 charge states often dominate for larger peptides.

Low-intensity peaks below 5% of base peak might represent impurities, fragments, or adducts. Adducts form when peptides bind to sodium (M+Na), potassium (M+K), or ammonium (M+NH4) instead of protons. These appear as peaks shifted by 22 Da (Na), 38 Da (K), or 17 Da (NH4) from the expected mass.

Expected vs Observed Mass

Calculating expected mass allows you to verify the peptide is what the vendor claims.

Calculating Theoretical Mass

Sum the monoisotopic masses of all amino acids, subtract water molecules lost during peptide bond formation, and add masses of any modifications.

For unmodified peptides: Theoretical Mass = Sum of amino acid residue masses + 18.015 Da (N and C termini).

The 18.015 Da accounts for the H on the N-terminus and OH on the C-terminus that aren't removed during peptide bond formation.

Common amino acid monoisotopic masses (residue weights):

• Alanine (A): 71.037 Da
• Cysteine (C): 103.009 Da
• Aspartic acid (D): 115.027 Da
• Glutamic acid (E): 129.043 Da
• Phenylalanine (F): 147.068 Da
• Glycine (G): 57.021 Da
• Histidine (H): 137.059 Da
• Isoleucine (I): 113.084 Da
• Lysine (K): 128.095 Da
• Leucine (L): 113.084 Da
• Methionine (M): 131.040 Da
• Asparagine (N): 114.043 Da
• Proline (P): 97.053 Da
• Glutamine (Q): 128.059 Da
• Arginine (R): 156.101 Da
• Serine (S): 87.032 Da
• Threonine (T): 101.048 Da
• Valine (V): 99.068 Da
• Tryptophan (W): 186.079 Da
• Tyrosine (Y): 163.063 Da

For the peptide GHRP-6 (His-D-Trp-Ala-Trp-D-Phe-Lys):

137.059 + 186.079 + 71.037 + 186.079 + 147.068 + 128.095 + 18.015 = 873.432 Da

The D-amino acids (D-Trp, D-Phe) have identical masses to their L-forms. Mass spectrometry cannot distinguish stereoisomers.

Accounting for Modifications

Modifications alter mass predictably. Acetylation (N-terminus or lysine side chain) adds 42.011 Da. Amidation (C-terminus) subtracts 0.984 Da. Disulfide bonds subtract 2.016 Da (two hydrogens lost per bond). Phosphorylation adds 79.966 Da.

For acetyl-GHRP-6: 873.432 + 42.011 = 915.443 Da.

Always verify modifications are listed on the CoA. A mass discrepancy of 42 Da suggests unwanted acetylation occurred during synthesis.

Error Tolerance and Acceptance Criteria

No measurement is perfectly accurate. The acceptable error depends on the mass spectrometer type and the peptide molecular weight.

Instrument Resolution and Accuracy

Low-resolution instruments (single quadrupole, ion trap) provide accuracy around 0.1-0.5 Da or 100-500 ppm (parts per million). These are adequate for confirming identity but may miss small impurities.

High-resolution instruments (TOF, Orbitrap, FTICR) provide accuracy under 0.01 Da or below 10 ppm. These can distinguish between compounds differing by single Da and confidently identify molecular formulas.

Calculate ppm error using: ppm error = [(Observed Mass - Theoretical Mass) / Theoretical Mass] × 1,000,000.

For a peptide with theoretical mass 873.432 Da and observed mass 873.456 Da: ppm error = [(873.456 - 873.432) / 873.432] × 1,000,000 = 27.5 ppm.

Acceptable Error Ranges

For peptides under 2000 Da analyzed on high-resolution instruments, expect error under 20 ppm or 0.02 Da. For peptides 2000-5000 Da, accept error under 50 ppm or 0.1 Da. For peptides over 5000 Da, error under 100 ppm or 0.2 Da is reasonable.

Low-resolution data should fall within 0.3 Da for small peptides and 0.5 Da for large peptides.

If observed mass differs by more than 1 Da from expected, the peptide is likely not what was claimed. A difference of exactly 14 Da suggests an extra methylene group (wrong amino acid). A difference of 16 Da suggests oxidation, often affecting methionine or tryptophan. A difference of 32 Da suggests double oxidation.

Why MS and HPLC Together

Mass spectrometry and HPLC (High-Performance Liquid Chromatography) provide complementary information. Using both is standard practice for peptide verification.

HPLC Measures Purity

HPLC separates compounds based on hydrophobicity and measures what percentage of the sample is the target peptide versus impurities. A peak at the correct retention time representing 95% of total UV absorbance indicates 95% purity.

However, HPLC cannot confirm identity. A peak at 8.5 minutes could be your target peptide or a different compound with similar hydrophobicity. Deletion sequences (missing one amino acid) or substitution sequences (wrong amino acid) often have similar retention times.

MS Confirms Identity

Mass spectrometry confirms molecular weight but is poor at quantifying purity. Ion suppression effects mean impurities may ionize more or less efficiently than the target peptide. A small impurity with high ionization efficiency can produce a peak similar in intensity to the target. A large impurity with poor ionization efficiency might barely appear.

Additionally, MS shows what masses are present but not what percentage of the total sample each represents without quantitative standards.

LC-MS Combines Both

LC-MS (liquid chromatography-mass spectrometry) runs HPLC separation followed by direct injection into the mass spectrometer. This provides retention time, purity estimate, and mass confirmation simultaneously.

The chromatogram shows peaks over time. The mass spectrum shows the mass of compounds eluting at each time point. You can extract the mass spectrum at the retention time of the main HPLC peak to confirm that peak is your target peptide.

If the main HPLC peak shows the expected mass, you have both purity and identity confirmation. If the main peak shows an unexpected mass, the peptide is mislabeled. If multiple HPLC peaks show masses close to the target, you have a mixture of related peptides (deletion sequences, oxidation variants).

Red Flags in MS Data

Certain patterns indicate problems with synthesis, handling, or data authenticity.

Missing Charge States

Peptides should show multiple charge states. A spectrum showing only a single m/z peak without isotope pattern or additional charge states suggests fabricated data or poor ionization conditions.

For peptides 1000-3000 Da, expect at least two charge states (+2 and +3). Seeing only one suggests the spectrum was faked or the instrument settings were incorrect.

Wrong Isotope Patterns

The isotope spacing and intensity ratio must match the charge state and molecular size. Isotope peaks separated by 1.0 m/z for a claimed +2 charge state is physically impossible.

A small peptide (500 Da) showing an M+1 peak at 80% of M peak intensity suggests the data is fabricated. The M+1 should be under 20% for this size.

Suspiciously Perfect Mass Match

Observed mass matching theoretical mass to four decimal places (873.4321 observed vs 873.4321 theoretical) is statistically unlikely. High-resolution instruments have accuracy limits. Expect some deviation, even if small.

A series of CoAs from the same vendor all showing observed mass matching theoretical mass to three or more decimal places suggests numbers were copied from theoretical calculations rather than measured.

Excessive Impurity Peaks

More than 3-5 significant impurity peaks (over 10% of base peak intensity) indicates poor synthesis or degradation. Common impurities include deletion sequences (n-1, n-2 peptides missing amino acids), oxidation products (+16 or +32 Da), and trifluoroacetate adducts (+114 Da).

If impurity peaks are more intense than the target peptide peak, the product purity is below 50%. This is unacceptable for research-grade peptides.

Unrealistic Baseline

The baseline should show low-level noise. A perfectly flat baseline at exactly zero intensity across the entire spectrum suggests the data was manually created or heavily processed. Real instrument noise produces small fluctuations.

Generic or Recycled Spectra

Some vendors use the same MS spectrum for multiple similar peptides. Compare spectra from different orders. If the mass changes but the isotope pattern, relative peak intensities, and noise pattern remain identical, the vendor is fabricating data.

Request raw data files (.RAW, .WIFF, .D) if you suspect fraud. These contain metadata including acquisition date, instrument serial number, and method parameters that are difficult to fake.

Spotting Fake MS Results

The underground peptide market is rife with falsified Certificates of Analysis. Vendors copy spectra from legitimate sources or generate fake data using software.

Data Without Context

Legitimate MS data includes instrument type, acquisition date, operator information, and method parameters. A spectrum showing only m/z and intensity without metadata is suspicious.

The CoA should state the instrument model (e.g., "Agilent 6545 Q-TOF", "Thermo Q Exactive Orbitrap"). If only "mass spectrometry" is listed without instrument details, verify carefully.

Perfect Peak Symmetry

Real peaks have slight asymmetry from tailing effects and imperfect ion focusing. Peaks that are perfectly Gaussian or perfectly symmetrical were likely generated with graphing software.

Look at the peak shape. Real peaks from ESI-MS show slight fronting (sharper leading edge) or tailing (sharper trailing edge). Synthetic peaks are often too perfect.

Image-Only Data

Legitimate labs provide spectral data as PDFs exported from instrument software or as raw data files. A spectrum provided as a JPEG or low-resolution screenshot suggests it was copied from another source or created in Photoshop.

Request a high-resolution PDF or the raw data file. Vendors with nothing to hide will provide it. Vendors fabricating data will make excuses.

Impossible Mass Values

Calculate expected mass independently and compare. If the vendor claims a mass that differs from your calculation by more than 1 Da (excluding modifications you may have missed), the data is wrong or the sequence is wrong.

Some vendors intentionally mislabel peptides. They synthesize a cheap peptide and claim it's an expensive one, providing fake MS data showing the expected mass of the expensive peptide.

Batch Number Inconsistencies

If you order the same peptide multiple times, each batch should have unique MS data showing slight variations in impurity profiles. Vendors providing identical spectra with different batch numbers are reusing data.

Note minor differences in impurity peaks between batches. These should vary slightly. If the main peak and every single impurity peak have identical m/z and intensity across multiple batches, the data was copied.

Cross-Reference with Known Spectra

For common peptides, spectra may be published in literature or peptide databases. A reverse image search can reveal if a vendor copied a spectrum from a published source.

The PeptideAtlas and PRIDE databases contain experimental MS/MS spectra for thousands of peptides. If your vendor's spectrum matches a published spectrum from a different lab with identical noise patterns, it's stolen.

Request Third-Party Testing

When in doubt, send a sample to an independent testing lab. Several companies provide peptide analysis services for $100-300. Compare their results to the vendor's CoA.

If the third-party testing shows a different mass or significantly lower purity, the vendor's CoA was fabricated. Document this and report to peptide community forums to warn others.

Conclusion

Mass spectrometry provides definitive confirmation of peptide identity when interpreted correctly. Understanding charge states, isotope patterns, mass accuracy, and common artifacts allows you to verify analytical data and detect fraudulent CoAs.

Always calculate expected mass independently. Verify observed mass falls within acceptable error for the instrument type. Check for multiple charge states and correct isotope patterns. Request raw data files when authenticity is questionable.

Combined HPLC-MS analysis provides both purity and identity confirmation. Neither technique alone is sufficient. Vendors providing only MS data without chromatographic purity or only HPLC purity without mass confirmation are cutting corners.

The research peptide market contains significant fraud. Developing the skills to critically evaluate MS data protects your research and your budget. Trust but verify. Always verify.