An evaluation of the utility of in vacuo methylation for mass-spectrometry-based analyses of peptides

In vacuo trimethylation of the N-terminus of a lyophilized peptide with methyl iodide was previously reported to enhance the peptide's signal in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and to suppress alkali adduct formation in electrospray ionization mass spectrometry (ESI-MS). Both the signal enhancement and alkali adduct suppression observed for methylated peptides are believed to be due to the permanent positive charge on the N-terminus of the peptide resulting from the formation of a quaternary ammonium moiety. The present work evaluates the general utility of the in vacuo methylation procedure for the MS analysis of peptides, and specifically addresses the issue of whether the methylation of nucleophilic sites other than the N-terminal amine affects the MALDI signal of modified peptides. This work establishes that, although certain side-chain modifications are inevitable using present reaction conditions, the derivatization leads to significant MALDI-MS signal improvement. The experimental results demonstrate that the N-terminal trimethylammonium derivatives of peptides exhibit MALDI signals comparable to or exceeding those of arginine-containing standards such as angiotensin I. The advantages and limitations of the in vacuo methylation procedure are discussed.     

Nanodiamond MALDI support for enhancing the credibility of identifying proteins

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a standard analytical tool for protein identification and peptide sequencing. High sensitivity and resolution are two critical parameters for recording good peptide mass fingerprinting (PMF) of low abundance proteins. Here, we report a novel nanodiamond (ND) (normal size 3–10 nm) support for MALDI-MS target, over which -cyano-4-hydrocinnamic acid (CCA) crystallizes evenly. Good reproducibility of relative peak intensity (R.S.D. less than 11.8%) among sample spot (from ring to center) is achieved on ND support. Therefore, the search for “hot spots” during the analysis is not necessary, which is supporting for the automatic acquisition of data. Due to high absorbability of energy from the laser, the ND support improves ionization efficiency of samples. In general, the sensitivity of MS obtained on ND support can be enhanced three to four times compared to the conventional MALDI sample preparation technique. Sensitivity obtained on ND support ranges from 62.5 amol of Arg-vasopressin standard peptide to 1.0 fmol of myoglobin tryptic peptide mixture. Reduced spot size and increased sensitivity in MALDI-MS are also accomplished by ND support. With spot size reduced, the signal intensity of cytochrome c (Cyt c) tryptic peptide obtained on ND support is at least seven times greater than it acquired on stainless steel. And ND support has been found better tolerance for salt (up to 500 mM NaCl) to MALDI-MS analysis. All these properties make ND support a valuable tool for MALDI-MS identification of proteins.     

C-Terminal sequencing of protein by MALDI mass spectrometry through the specific derivatization of the α-carboxyl group with 3-aminopropyltris-(2,4,6-trimethoxyphenyl)phosphonium bromide

We present here an approach to C-terminal sequencing of proteins by the procedure consisting of the following: (1) derivatization of the C-terminal α-carboxyl group with 3-aminopropyltris(2,4,6-trimethoxyphenyl)-phosphonium bromide (TMPP-propylamine) through oxazolone chemistry, (2) enzymatic proteolysis of the TMPP-derivatized protein, and (3) MALDI-MS/MS analysis of the peptide mixture, in which the C-terminal peptide incorporating the TMPP group is preferably detected. In this protocol, it is possible to choose any endoproteinase such as trypsin, GluC, and AspN for digestion so that a C-terminal peptide with length appropriate for mass spectrometric sequencing could be generated. The peptide labeled with TMPP-propylamine at the C terminus tends to exhibit y-type ions in MS/MS spectra, allowing manual sequence interpretation with the simplified fragmentation pattern. The efficacy of the method was verified with five proteins, which demonstrated that the C-terminal peptides were readily distinguishable by their peak intensity and characteristic mass signature peak in MALDI-PSD analysis.     

Diagnostic protein discovery using proteolytic peptide targeting and identification

Plasma protein profiling with mass spectrometry is currently being evaluated as a diagnostic tool for cancer and other diseases. These experiments consist of three steps: plasma protein fractionation, analysis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), and comparisons of the MALDI profiles to develop diagnostic fingerprints using bioinformatic techniques. While preliminary results appear promising in small sample groups, the method is limited by the sensitivity of MALDI-MS for intact proteins, the limited mass range of MALDI-MS, and difficulties associated with isolating individual proteins for identification to validate the diagnostic fingerprint. Here we present an alternative and improved method directed toward diagnostic protein discovery, which incorporates proteolytic peptide profiling, bioinformatic targeting of ion signals, and MALDI tandem mass spectrometry (MS/MS) peptide sequencing, rather than fingerprinting. Pancreatic cancer patients, pancreatitis patients, and controls are used as the model system. Profiling peptides after enzymatic digestion improves sensitivity and extends the accessible protein molecular weight range when compared to intact protein profiling. The first step is to extract and fractionate the proteins from plasma. Each fraction is digested with trypsin and subsequently analyzed by MALDI-MS. Rather than using bioinformatic analysis as a pattern-matching technique, peptides are targeted based on the disease to control peak intensity ratios measured in the averages of all mass spectra in each group and t-tests of the intensity of each individual peak. The targeted peptide ion signals are subsequently identified using MALDI-MS/MS in quadrupole-TOF and tandem-TOF instruments. This study found not only the proteins targeted and identified by a previous protein profiling experiment, but also detected additional proteins. These initial results are consistent with the known biology of pancreatic cancer or pancreatitis, but are not specific to those diseases.     

Effects of common surfactants on protein digestion and matrix-assisted laser desorption/ionization mass spectrometric analysis of the digested peptides using two-layer sample preparation

While surfactants are commonly used in preparing protein samples, their presence in a protein sample can potentially affect the enzymatic digestion process and the subsequent analysis of the resulting peptides by mass spectrometry. The extent of the tolerance of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to surfactant interference in peptide analysis is very much dependent on the matrix/sample preparation method. In this work the effects of four commonly used surfactants, namely n-octyl glucoside (OG), Triton X-100 (TX-100), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and sodium dodecyl sulfate (SDS), for biological sample preparation on trypsin digestion and MALDI-MS of the resulting digest are examined in detail within the context of using a two-layer method for MALDI matrix/sample preparation. Non-ionic and mild surfactants, such as OG, TX-100 or CHAPS, are found to have no significant effect on trypsin digestion with surfactant concentrations up to 1%. However, TX-100 and CHAPS interfere with the subsequent peptide analysis by MALDI-MS and should be removed prior to peptide analysis. OG is an MS-friendly surfactant and no effect is observed for MALDI peptide analysis. The effect of SDS on trypsin digestion in terms of the number of peptides generated and the overall protein sequence coverage by these peptides is found to be protein dependent. The use of SDS to solubilize hydrophobic membrane proteins, followed by trypsin digestion in the presence of 0.1% SDS, results in a peptide mixture that can be analyzed directly by MALDI-MS. These peptides are shown to provide better sequence coverage compared with those obtained without the use of SDS in the case of bacteriorhodopsin, a very hydrophobic transmembrane protein. This work illustrates that MALDI-MS with the two-layer sample preparation method can be used for direct analysis of protein digests with no or minimum sample cleanup after proteins are digested in a solution containing surfactants.     

Synthesis of functionalized guanidino amino acids

We report the synthesis of guanidino amino acids (GuAA), which are structurally related to Arg and resemble a dipeptide consisting of alpha- and gamma-amino acid with a guanidinium group in the main chain. The compounds are available with different protecting groups in gram amounts and are intended as synthetic building blocks for the construction of synthetic oxoanion or peptide receptors. Tyr, Trp or dansyl-functionalized Lys can be introduced as the alpha-amino acid part, which leads to luminescent GuAAs. The compounds signal carboxylate binding in MeOH, DMSO and buffered water by change of the emission intensity. The property may find use in the construction of chemosensors.     

Isotope edited product ion assignment by α-N labeling of peptides with [2H3(50%)]2,4-Dinitrofluorobenzene

An isotopic modification of Sanger's method for identifying peptide N-termini has been developed to assist peptide sequencing by tandem mass spectrometry. Tryptic peptides, such as Val-His-Leu-Thr-Pro-Val-Glu-Lys, are derivatized with an equimolar mixture of 2,4-dinitrofluorobenzene and [2H3]2,4-dinitrofluorobenzene. Under optimized derivatization conditions, the alpha-amino group could be derivatized while the epsilon-amine of the lysine side chain and the imidazole of histidine remained underivatized. The alpha-dinitrophenyl modified peptides were characterized by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) and liquid chromatography (LC)-ESI-MS. The [M + H]+ ions showed a doublet pattern with a delta m/z of 3 and the [M + 2H]2+ ions were recognized as doublets with a delta m/z of 1.5. MS/MS was employed where both isotopic [M + 2H]2+ ions were alternately subjected to collision-induced dissociation in the second quadrupole. Fragmentation in the ionization source generated identical product ion patterns that were observed during fragmentation in the second quadrupole. In the product ion mass spectra, the N-terminal a and b ions (no c ion observed) are doublets with a delta m/z of 3 or 1.5, while the C-terminal y and z ions (no x ion observed) are singlets appearing at identical masses. Thus, the product ions containing the N-terminus derivatized with a dinitrophenyl group are unequivocally distinguished from the product ions containing the C-terminus. The dinitrophenyl modification generally enhanced the production of a and b ions without diminishing y and z ion yields.     

Improving the signal intensity and sensitivity of MALDI mass spectrometry by using nanoliter spots deposited by induction-based fluidics

A new contact-free, small droplet deposition method using an induction-based fluidics (IBF) technique to dispense nanoliter drops is described and evaluated for sample preparation in matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The signal intensities available when using nanoliter spots are greater than those obtained with normal, microliter spots when the same amount of analyte is used. When using an ionic-liquid matrix, the improvement in sensitivity is equal to the concentration enhancement that was achieved by using smaller quantities of matrix. When using a conventional solid matrix, however, the increase in signal intensity shows a more complicated relationship to concentration. The approach of nanoliter deposition also supports multiple spotting to increase sample concentration and, thus, sample signal intensity. Nanoliter spotting not only improves the signal intensity and sensitivity achieved by MALDI-MS but also allows a major fraction of trace samples to be saved for other experiments, thus expanding the application of MALDI-MS to biological studies where sample quantity is limited.     

Phosphoric acid enhances the performance of Fe(III) affinity chromatography and matrix-assisted laser desorption/ionization tandem mass spectrometry for recovery, detection and sequencing of phosphopeptides

An integrated analytical strategy for enrichment, detection and sequencing of phosphorylated peptides by matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS) is reported. o-Phosphoric acid was found to enhance phosphopeptide ion signals in MALDI-MS when used as the acid dopant in 2,5-dihydroxybenzoic acid (2,5-DHB) matrix. The effect was largest for multiply phosphorylated peptides, which exhibited an up to ten-fold increase in ion intensity as compared with standard sample preparation methods. The enhanced phosphopeptide response was observed during MALDI-MS analysis of several peptide mixtures derived by proteolytic digestion of phosphoproteins. Furthermore, the mixture of 2,5-DHB and o-phosphoric acid was an excellent eluant for immobilized metal affinity chromatography (IMAC). Singly and multiply phosphorylated peptide species were efficiently recovered from Fe(III)-IMAC columns, reducing sample handling for phosphopeptide mapping by MALDI-MS and subsequent phosphopeptide sequencing by MALDI-MS/MS. The enhanced response of phosphopeptide ions in MALDI facilitates MS/MS of large (>3 kDa) multiply phosphorylated peptide species and reduces the amount of analyte needed for complete characterization of phosphoproteins.     

Surface-assisted laser desorption-ionization mass spectrometry on titanium dioxide (TiO2) nanotube layers

The paper reports on the use of a titanium oxide (TiO(2)) nanotube layer as a sensitive substrate for surface-assisted laser desorption-ionization mass spectrometry (SALDI-MS) of peptides and small molecules. The nanotube layers were prepared by electrochemical anodization of titanium foil. The optimized TiO(2) nanotubes morphology coupled to a controlled surface chemistry allowed desorption-ionization (D/I) of a peptide mixture (Mix1) with a detection limit of 10 femtomoles for the neurotensin peptide. The performance of the TiO(2) nanotubes for the D/I of small molecules was also tested for the detection of sutent, a small tyrosine kinase inhibitor, and verapamil. A detection limit of 50 fmol was obtained for these molecules, as compared to 500 fmol using classical matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). Both amorphous and anatase TiO(2) layers displayed a comparable performance for D/I of analyte molecules. In a control experiment, we have performed D/I of analyte molecules on a flat TiO(2) layer. The absence of signal emphasizes the role of the nanostructured substrate in the D/I process.     

Ionization and dissociation of CH3I in intense laser field

The ionization-dissociation of methyl iodide in intense laser field has been studied using a reflection time-of-flight mass spectrometry (RTOF-MS), at a laser intensity of < or =6.6x10(14) W/cm(2), lambda=798 nm, and a pulse width of 180 fs. With the high resolution of RTOF-MS, the fragment ions with the same M/z but from different dissociation channels are resolved in the mass spectra, and the kinetic energy releases (KERs) of the fragment ions such as I(q+) (q=1-6), CH(m) (+) (m=0-3), C(2+), and C(3+) are measured. It is found that the KERs of the fragment ions are independent of the laser intensity. The fragments CH(3) (+) and I(+) with very low KERs (<1 eV for CH(3) (+) and <0.07 eV for I(+)) are assigned to be produced by the multiphoton dissociation of CH(3)I(+). For the fragments CH(3) (+) and I(+) from CH(3)I(2+), they are produced by the Coulomb explosion of CH(3)I(2+) with the interaction from the covalent force of the remaining valence electrons. The split of the KER of the fragments produced from CH(3)I(2+) dissociation is observed experimentally and explained with the energy split of I(+)((3)P(2)) and I(+)((3)P(0,1)). The dissociation CH(3)I(3+)-->CH(3) (+)+I(2+) is caused by Coulomb explosion. The valid charge distance R(c) between I(2+) and CH(3) (+), at which enhanced ionization of methyl iodide occurs, is obtained to be 3.7 A by the measurements of the KERs of the fragments CH(3) (+) and I(2+). For the CH(3)I(n+) (n> or =3), the KERs of the fragment ions CH(3) (p+) and I(q+) are attributed to the Coulomb repulsion between CH(3) (p+) and I(q+) from R(c) approximately 3.7 A. The dissociation of the fragment CH(3) (+) is also discussed. By the enhanced ionization mechanism and using the measured KER of I(q+), all the possible Coulomb explosion channels are identified. By comparing the abundance of fragment ions in mass spectrum, it is found that the asymmetric dissociation channels with more charges on iodine, q>p, are the dominant channels.     

Photoionization of CH(3)I mediated by the C state in the visible and ultraviolet regions

Three/two-photon resonant multiphoton ionization (MPI) of the CH3I monomer has been studied in the gas phase at 532 and 355 nm using time-of-flight mass spectrometry. Under low laser intensity (approximately 10(9) W/cm2) the mass spectra showed peaks at m/z 15, 127 and 142, corresponding to [CH3]+, [I]+ and [CH3I]+ species, at both these wavelengths. The laser power dependence for [CH3I]+, [I]+ and [CH3]+ ions showed a three-photon dependence at 532 nm. For the same three ions, photoionization studies at 355 nm gave a power dependence of 2. Both these results suggest that a vibronic energy level at approximately 7 eV, lying in the Rydberg C state, acts as a resonant intermediate level in ionization of CH3I. In the case of 355 nm, with increasing intensity additional peaks at m/z 139 and 141 were observed which could be assigned to [CI]+ and [CH2I]+ fragments. In contrast, for high intensity radiation at 532 nm ( approximately 2 x 10(10) W/cm2), only the [CI]+ fragment was observed. At these wavelengths, fragment ions observed in mass spectra mainly arise from photodissociation of the parent ion. Experiments at another wavelength in the visible region (564.2 nm) confirmed the results obtained at 532 nm. In order to assess the role of the A state in these MPI experiments, additional experiments were performed at 266 and 282.1 nm, which access the A state directly via a one-photon transition, and showed absence of a surviving precursor ion. Reaction energies for various possible dissociation channels of CH3I/[CH3I]+/[CH2I]+ were calculated theoretically at the MP2 level using the GAMESS electronic structure program.     

The contributions of specific amino acid side chains to signal intensities of peptides in matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of complex peptide mixtures is often hampered by signal suppression effects as well as certain intrinsic properties of specific peptides that influence the desorption/ionization behavior. The present systematic study reports on the relationship between the occurrence of certain amino acids in peptides and the intensities of the related ions which appear during MALDI-MS analysis for both tryptic digests of proteins and synthetic peptide mixtures. The analysis of the tryptic digests revealed that the peptide sequences of the most intense peaks detected by MALDI-MS contained significantly higher proportions of arginine, phenylalanine, proline, and leucine than the average values for the measured proteins. The relationship between the relative signal intensities and amino acid compositions of peptides was studied in more detail by the partial least squares (PLS) method using equimolar mixtures of 144 well-characterized synthetic peptides. The regression coefficients clearly indicated that the presence of arginine, phenylalanine, leucine and proline tend to enhance the desorption/ionization process which results in higher MALDI-MS peak intensities. Furthermore, it was shown that the impact of arginine depends strongly on the identity of adjacent amino acids.     

Hydrogen-deuterium exchange and selective labeling of deprotonated amino acids and peptides in the gas phase

Hydrogen-deuterium exchange reactions of deprotonated amino acids and small peptides were studied. Selective labeling can be carried out at the alpha-amino group of lysine (2 of 4 labile hydrogens undergo exchange with CF3CH2OD) and the guanidine side chain of arginine (3 of 6 labile hydrogens undergo exchange with CH3CH2OD and C6H5CH2OD). Differential labeling of peptides also was accomplished, and the extent of H/D exchange is dependent on the amino acids which are present as well as their order in the chain.     

Enhanced responses in matrix-assisted laser desorption/ionization mass spectrometry of peptides derivatized with arginine via a C-terminal oxazolone

We have developed a novel method for enhancing the response of a peptide in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) by activating the C-terminal carboxyl group through an oxazolone with which is coupled an amine containing a functional group to help ionize the peptide. The reactions consist of dehydration with acetic anhydride to give an oxazolone, followed by aminolysis with an appropriate amino acid derivative such as arginine methyl ester. The MALDI signal of Ac-Tyr-Gly-Gly-Phe-Leu-Arg-OMe, thus converted from leucine-enkephalin, was detected while completely excluding the responses of arginine-deficient peptides coexisting in the reaction mixture. Some less intense peaks corresponding to a few sequential degradation products, also terminated with the arginine derivative, were also observed. The side-chain groups potentially that are reactive were conveniently protected by acetylation simultaneous with the C-terminal activation, and those that remained unprotected were reduced to virtually negligible proportions when the reaction was conducted in a peptide solution of concentration less than 1 mM. The greatly increased responses of such arginine-terminated peptides could possibly be exploited to discern the C-terminal tryptic peptide of a protein that is otherwise almost insensitive to MALDI-MS in general. The simplicity of the post-source decay spectrum of enkephalin derivatized by arginine methyl ester characteristically accentuated z- and b-type ions, and this should facilitate sequencing of such derivatized peptides. Remaining problems with practical applications of this approach are discussed.     

Sub-microlitre dialysis system to enable trace level peptide detection from volume-limited biological samples using MALDI-TOF-MS

The detection of peptides with mass spectrometry from volume-limited biological samples is a challenging task due to low sample volume, a broad range of peptide concentrations down to trace levels, endogenous high proteins and salt levels. Previously, a microspotting method was presented for trace-level peptide detection with MALDI-MS from sub-microlitre samples with biological salt levels. However, in the presence of proteins, peptide signals are significantly reduced. This paper presents a novel dialysis device for removal of proteins from sub-microlitre samples using a semipermeable hollow fiber membrane to enhance peptide detection. A dialysis device was constructed to perform sub-microlitre dialysis to remove proteins from complex samples. Angiotensin I was used as a model peptide in the presence of 350 mg L(-1) BSA prepared in physiological saline to mimic biological samples. In the absence of BSA, clear angiotensin I peaks were seen at 250 pM, yet in the presence of the BSA, 10 nM angiotensin I was barely detected. After dialysis, peak detection was improved to a 500 pM level. Protein removal and peptide recovery (approximately 66%) were determined using CE-LIF. Clinical vitreous samples as low as 200 nL were successfully dialyzed in 30 min and a 3-fold increase in peptide peaks were detected with greatly improved signals. This method is simple and can be a useful technique for trace level peptide detection from volume-limited biological samples.     

Characterization of polyether mixtures using thin-layer chromatography and matrix-assisted laser desorption/ionization mass spectrometry

Thin-layer chromatography (TLC) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) were combined to achieve characterization of polyether mixtures. Three polyethers, polyethylene glycol (PEG), polypropylene glycol (PPG) and polytetramethylene glycol (PTMG), or mixtures of these compounds, were studied. One shortcoming of mixture analysis of synthetic polymers using MALDI-MS is that individual polymers in the mixture may display different detection sensitivities. For example, the MALDI mass spectrum of an equimolar mixture of PEG, PPG and PTMG displayed a high intensity of PPG ions, while no PTMG ions were detectable; however, PTMG ions were detected after the mixture had been separated by TLC. This combined TLC and MALDI-MS analysis of a PPG polymer bearing reactive epoxy groups showed that the polymer contained byproducts with different end-groups. These byproducts were identified as chloro-substituted polymers formed during polymer synthesis. Our study shows TLC to be a rapid and low-cost separation technique, and that it can be combined with MALDI-MS to achieve effective analysis of synthetic polymers.     

Ion-pair assisted recovery of matrix-assisted laser desorption/ionization mass spectral signals from SDS-containing peptide-protein mixtures

We have developed a simple and effective means of using alkylammonium ion-pairing agents, such as cetyltetramethylammonium bromide, to recover matrix assisted laser desorption/ionization mass spectrometric (MALDI-MS) signals from sodium dodecyl sulfate (SDS)-containing protein and peptide samples. A two-layer method of matrix preparation, with a bottom matrix layer of ion-pairing agents and a top matrix layer of SDS protein samples, is essential for reproducible MALDI mass spectra with good recovery. Both buffer ions and ion-pairing agents have profound effects on signal recovery and can be rapidly and systematically optimized. This practical technique, termed ion-pair assisted recovery (IPAR), is compatible with major SDS-based biotechniques and can be easily incorporated into high-throughput proteomic analysis.     

Epsilon -N,N,N-trimethyllysine-specific ions in matrix-assisted laser desorption/ionization-tandem mass spectrometry

Epsilon -N,N,N-trimethyllysine (K(me3)) is a component of a number of proteins and plays an important role in the expression of their biological functions. Trimethylation, which causes an incremental increase in mass of 42.0470 Da from that of the corresponding MH(+) ion, cannot be distinguished from the acetylation (+42.0106 Da), which also occurs on epsilon-amino groups of Lys or alpha-amino groups in many proteins, without high-accuracy mass measurement which is accurate to within the second decimal place. MALDI-MS and MS/MS have been applied for the analyses of post-translational modifications of histone H3, which is known to contain both multiple acetylation and methylation sites in its sequence. During the measurements of the modified peptides, a novel fragmentation which involves the loss of trimethylamine from K(me3) was found. This characteristic fragmentation, which was observed to produce ions separated by 59 Da from the conventional precursor ion or sequence ions, would be useful for probing K(me3) units in the sequence.     

MALDI-MS analysis of peptides modified with photolabile arylazido groups

The ability of MALDI-MS to analyze photolabile arylazido peptide derivatives was investigated. Peptides containing UV-labile p-azidobenzoyl groups were subjected to MALDI-MS analysis in a variety of matrices. As standard MALDI-MS employs a UV laser (337 nm), we investigated conditions that would allow detection of the intact molecule ions for these light-sensitive peptides. When using α-cyano-4-hydroxycinnamic acid (ACHC) or 2,5 dihydroxybenzoic acid (DHB) as the matrix, photoinduced degradation products were prevalent. In contrast, when employing the matrix sinapinic acid, the intact molecule ion corresponding with the azido peptide was the predominant signal. The protection of photolabile azido derivatives correlates with the UV absorbance properties of the matrix employed, i.e., sinapinic acid, which exhibits a strong absorbance near 337 nm, most efficiently protects the azido derivative from photodegradation.