Dipartimento di Chimica, Università della Calabria, Arcavacata di Rende, Italy.
Anal Chem. 2010 Jul 1;82(13):5552-60. doi: 10.1021/ac1004212.
The solid phase isobaric mass tagging (SPIMT) approach is presented for simultaneous protein quantitation and identification. The novelty of the SPIMT strategy relies on a CID-based differentiation of regioisomeric species for quantitation of tagged proteolytic peptides. SPIMTs are unlabeled mass-tagging reagents, which consist of a reporter group, a mass balance group, and a spacer with a amine-specific reactive group, able to be linked to any N-terminal peptide. Therefore SPIMT-linked peptides from a two-plex set appear as a single unresolved precursor ion in MS, whereas the reporter groups lead to quantitation signals of m/z 168.2 and 182.2 Da upon tandem mass spectrometry (MS/MS) analysis with matrix-assisted laser desorption time-of-flight/time-of-flight (MALDI TOF/TOF). This strategy allows ease protein identification by direct submission of MS and MS/MS data to the MASCOT database. SPIMT approach showed an excellent quantitation linearity, detecting any relative concentration differences of peptides in two solutions over a 5-fold concentration range without losing sequencing information. Therefore, SPIMTs are an attractive, simple, and low cost alternative for two-plex quantitation of proteins and offer possibilities of tuning the two-plex signal mass window by replacing the spacer.
固相等压质量标记（SPIMT）方法被提出用于同时进行蛋白质定量和鉴定。SPIMT 策略的新颖之处在于基于 CID 的区域异构体的区分，用于定量标记的肽酶解肽。SPIMTs 是未标记的质量标记试剂，由报告基团、质量平衡基团和带有胺特异性反应基团的间隔基组成，能够与任何 N 端肽连接。因此，来自双相集的 SPIMT 连接肽在 MS 中表现为单个未解析的前体离子，而报告基团在基质辅助激光解吸飞行时间/飞行时间（MALDI TOF/TOF）串联质谱（MS/MS）分析中导致 m/z 168.2 和 182.2 Da 的定量信号。这种策略允许通过直接将 MS 和 MS/MS 数据提交到 MASCOT 数据库来轻松进行蛋白质鉴定。SPIMT 方法表现出出色的定量线性度，在不丢失测序信息的情况下，能够检测两种溶液中肽的任何相对浓度差异超过 5 倍的浓度范围。因此，SPIMTs 是一种有吸引力的、简单的、低成本的双相定量蛋白质替代方法，并提供通过替换间隔基来调整双相信号质量窗口的可能性。
